<p>Cervical contusion SCI was administered at the C5 or C6 level of the spinal cord while u... more <p>Cervical contusion SCI was administered at the C5 or C6 level of the spinal cord while uninjured control animals received only laminectomy at the C6 level. Tissue from the level of laminectomy or epicenter of the injury and from the region immediately caudal was harvested for either immunoblotting or histology (A–B). Thermal hyperalgesia was measured using a modified version of the Hargreaves test. Animals receiving contusion injury showed a decrease in latency to withdrawal in the ipsilateral forepaw, measured as a percentage of baseline latency, compared to control animals (C). This difference was first observed at two weeks after injury and persisted until the animals were sacrificed at six weeks after injury. No change in withdrawal latency was seen in injured vs. uninjured animals in the contralateral forepaw (D). Animals receiving SCI were also tested for tactile allodynia using von Frey filament testing. In animals receiving C6 injury, but not C5 injury, a significant decrease compared to pre-injury baseline in the force threshold required to elicit a withdrawal response was evident both ipsilaterally (E) and contralaterally (F) for each of the six weeks following injury. The decrease in ipsilateral forepaw withdrawal latency and bilateral force threshold in injured animals occurred in the absence of changes in grip strength in either forepaw (G–H). Epi = epicenter; IB = immunoblotting; histo = histology; * = p<0.05; ** = p<0.01; **** = p<0.0001.</p
<p>Astrocyte activation in the superficial dorsal horn was characterized by quantification ... more <p>Astrocyte activation in the superficial dorsal horn was characterized by quantification of GFAP expression in laminae I–II. (A) and (B) show representative images of dorsal horn GFAP expression at high magnification in laminectomy and injured animals, respectively. At the injury epicenter, compared to uninjured control animals (C), an increase in GFAP expression in the ipsilateral dorsal horn was evident at two (D) and six weeks (E) following injury. No significant changes in GFAP expression were observed at the other regions studied (F). IF = immunofluorescence; * = p<0.05; *** = p<0.001.</p
<p>Immunohistochemical analysis revealed a decrease in GLT1 protein expression two (B) and ... more <p>Immunohistochemical analysis revealed a decrease in GLT1 protein expression two (B) and six weeks (C) after cervical contusion SCI compared to laminectomy (A). This downregulation of the glutamate transporter was seen in the superficial dorsal horn on both sides of the spinal cord both at the injury site and caudal to the injury (D). Immunoblots of spinal cord tissue also showed a significant loss of GLT1 expression on the ipsilateral side at both time points following injury. However, this difference was seen only at the epicenter (E) and not caudal to the injury (F). Representative immunoblots are shown for each region (E–F). Analysis of GLT1 levels in regions of the spinal cord other than the superficial laminae at six weeks after injury reveal a loss of GLT1 expression in the ventral horn at the epicenter with no changes caudal to the injury (G). IF = immunofluorescence; epi = epicenter; ipsi = ipsilateral; contra = contralateral; lam = laminectomy; * = p<0.05; ** = p<0.01; *** = p 0.001.</p
<p>In the superficial laminae of the injured ipsilateral dorsal horn, we observed cells co-... more <p>In the superficial laminae of the injured ipsilateral dorsal horn, we observed cells co-expressing GFAP and Ki67 (A), representing activated and proliferating astrocytes. In laminectomy control animals, little to no cell proliferation was evident (B). However, the number of Ki67-positive cells was significantly increased at two weeks (C) and six weeks (D) after injury on the ipsilateral side both at the level of and caudal to the injury (E). Additionally, at two weeks, there was a significant increase in proliferating cells contralaterally at the injury site (E). At both two and six weeks, a significant percentage of Ki67-positive cells were also either GFAP-positive or CD11b-positive (F). Compared to laminectomy (G), the intensity of CD11b expression in the superficial dorsal horn was greater in animals with C6 SCI at both two (H–I) and six (I) weeks after injury. This increase was significant at the injury site both ipsilaterally and contralaterally, as well as caudal to the injury on the contralateral side (I). * = p<0.05; ** = p<0.01; **** = p<0.0001.</p
<p>Harvested tissue was stained with Cresyl violet and Eriochrome cyanine. At the level of ... more <p>Harvested tissue was stained with Cresyl violet and Eriochrome cyanine. At the level of the laminectomy, uninjured control animals (A) exhibited large motor neurons in the ventral horn (arrowheads). At six weeks post-injury, animals receiving unilateral C5 (B) or C6 (C) contusion SCI showed a loss of these motor neurons at the injury epicenter but not 1.0 mm caudal to the injury (D). The spread of ventral horn motor neuron loss was approximately 1.0 mm rostrally and 1.0 mm caudally (2.0 mm total) from the epicenter (E). Immunohistochemical analyses of the injury models were performed in laminae I–II and lamina III of the cervical spinal cord dorsal horn (F–G). Lam = laminectomy.</p
<p>Following cervical contusion SCI, GLT1 expression/promoter activity, represented by eGFP... more <p>Following cervical contusion SCI, GLT1 expression/promoter activity, represented by eGFP-positive cells, was decreased in the superficial dorsal horn. In laminectomy animals, there were low levels of GFAP, although many of the cells that were GFAP-positive also expressed GLT1 (A–B). Following injury, however, an increase in GFAP was observed, but fewer of these activated astrocytes co-expressed GLT1 (C). Laminectomy control animals (D) had greater numbers of eGFP-positive cells compared to animals that received contusion injury two days, two weeks, or six weeks (E) prior to analysis. This decrease in GLT1 expression was observed at the injury epicenter in both the ipsilateral (F) and contralateral (G) superficial laminae as well as caudal to the injury on the ipsilateral side (H). No change in the number of eGFP-positive cells was found caudal to the injury on the contralateral side (I). * = p<0.05; ** = p<0.01; *** = p 0.001.</p
<p>ΔfosB staining was used to measure the extent of persistent neuronal activation in the s... more <p>ΔfosB staining was used to measure the extent of persistent neuronal activation in the spinal cord. Following injury, ΔfosB-positive nuclei (arrowheads) were evident in laminae I–II (A–C). At all regions, laminectomy control animals (A) showed little-to-no ΔfosB expression (D). Two weeks after C6 injury (B), a significant increase in numbers of ΔfosB expressing cells in the superficial laminae of the ipsilateral dorsal horn was observed at the injury epicenter and caudal to the injury (D). ΔfosB levels were further increased at all regions in animals sacrificed six weeks post-injury (C, D). * = p<0.05; ** = p<0.01; *** = p 0.001; **** = p<0.0001.</p
Development of neuropathic pain occurs in a major portion of traumatic spinal cord injury (SCI) p... more Development of neuropathic pain occurs in a major portion of traumatic spinal cord injury (SCI) patients, resulting in debilitating and often long-term physical and psychological burdens. Following SCI, chronic dysregulation of extracellular glutamate homeostasis has been shown to play a key role in persistent central hyperexcitability of superficial dorsal horn neurons that mediate pain neurotransmission, leading to various forms of neuropathic pain. Astrocytes express the major CNS glutamate transporter, GLT1, which is responsible for the vast majority of functional glutamate uptake, particularly in the spinal cord. In our unilateral cervical contusion model of mouse SCI that is associated with ipsilateral forepaw heat hypersensititvity (a form of chronic at-level neuropathic pain-related behavior), we previously reported significant and long-lasting reductions in GLT1 expression and functional GLT1-mediated glutamate uptake in cervical spinal cord dorsal horn. To therapeutically ...
Transplantation-based replacement of lost and/or dysfunctional astrocytes is a promising therapy ... more Transplantation-based replacement of lost and/or dysfunctional astrocytes is a promising therapy for spinal cord injury (SCI) that has not been extensively explored, despite the integral roles played by astrocytes in the central nervous system (CNS). Induced pluripotent stem (iPS) cells are a clinically-relevant source of pluripotent cells that both avoid ethical issues of embryonic stem cells and allow for homogeneous derivation of mature cell types in large quantities, potentially in an autologous fashion. Despite their promise, the iPS cell field is in its infancy with respect to evaluating in vivo graft integration and therapeutic efficacy in SCI models. Astrocytes express the major glutamate transporter, GLT1, which is responsible for the vast majority of glutamate uptake in spinal cord. Following SCI, compromised GLT1 expression/function can increase susceptibility to excitotoxicity. We therefore evaluated intraspinal transplantation of human iPS cell-derived astrocytes (hIPSA...
Amyotrophic lateral sclerosis (ALS) is characterized by relatively rapid degeneration of both upp... more Amyotrophic lateral sclerosis (ALS) is characterized by relatively rapid degeneration of both upper and lower motor neurons, with death normally occurring 2-5years following diagnosis primarily due to respiratory paralysis resulting from phrenic motor neuron (PhMN) loss and consequent diaphragm denervation. In ALS, cellular abnormalities are not limited to MNs. For example, decreased levels and aberrant functioning of the major central nervous system (CNS) glutamate transporter, GLT1, occur in spinal cord and motor cortex astrocytes of both humans with ALS and in SOD1(G93A) rodents, a widely studied ALS animal model. This results in dysregulation of extracellular glutamate homeostasis and consequent glutamate excitotoxicity, a primary mechanism responsible for MN loss in ALS animal models and in the human disease. Given these observations of GLT1 dysfunction in areas of MN loss, as well as the importance of testing therapeutic strategies for preserving PhMNs in ALS, we evaluated int...
The Journal of neuroscience : the official journal of the Society for Neuroscience, Jan 21, 2015
Spinal cord injury (SCI) patients develop chronic pain involving poorly understood central and pe... more Spinal cord injury (SCI) patients develop chronic pain involving poorly understood central and peripheral mechanisms. Because dysregulation of the voltage-gated Kv3.4 channel has been implicated in the hyperexcitable state of dorsal root ganglion (DRG) neurons following direct injury of sensory nerves, we asked whether such a dysregulation also plays a role in SCI. Kv3.4 channels are expressed in DRG neurons, where they help regulate action potential (AP) repolarization in a manner that depends on the modulation of inactivation by protein kinase C (PKC)-dependent phosphorylation of the channel's inactivation domain. Here, we report that, 2 weeks after cervical hemicontusion SCI, injured rats exhibit contralateral hypersensitivity to stimuli accompanied by accentuated repetitive spiking in putative DRG nociceptors. Also in these neurons at 1 week after laminectomy and SCI, Kv3.4 channel inactivation is impaired compared with naive nonsurgical controls. At 2-6 weeks after laminect...
The Journal of neuroscience : the official journal of the Society for Neuroscience, Jan 28, 2014
A major portion of spinal cord injury (SCI) cases affect midcervical levels, the location of the ... more A major portion of spinal cord injury (SCI) cases affect midcervical levels, the location of the phrenic motor neuron (PhMN) pool that innervates the diaphragm. While initial trauma is uncontrollable, a valuable opportunity exists in the hours to days following SCI for preventing PhMN loss and consequent respiratory dysfunction that occurs during secondary degeneration. One of the primary causes of secondary injury is excitotoxic cell death due to dysregulation of extracellular glutamate homeostasis. GLT1, mainly expressed by astrocytes, is responsible for the vast majority of functional uptake of extracellular glutamate in the CNS, particularly in spinal cord. We found that, in bacterial artificial chromosome-GLT1-enhanced green fluorescent protein reporter mice following unilateral midcervical (C4) contusion SCI, numbers of GLT1-expressing astrocytes in ventral horn and total intraspinal GLT1 protein expression were reduced soon after injury and the decrease persisted for ≥6 weeks...
<p>Cervical contusion SCI was administered at the C5 or C6 level of the spinal cord while u... more <p>Cervical contusion SCI was administered at the C5 or C6 level of the spinal cord while uninjured control animals received only laminectomy at the C6 level. Tissue from the level of laminectomy or epicenter of the injury and from the region immediately caudal was harvested for either immunoblotting or histology (A–B). Thermal hyperalgesia was measured using a modified version of the Hargreaves test. Animals receiving contusion injury showed a decrease in latency to withdrawal in the ipsilateral forepaw, measured as a percentage of baseline latency, compared to control animals (C). This difference was first observed at two weeks after injury and persisted until the animals were sacrificed at six weeks after injury. No change in withdrawal latency was seen in injured vs. uninjured animals in the contralateral forepaw (D). Animals receiving SCI were also tested for tactile allodynia using von Frey filament testing. In animals receiving C6 injury, but not C5 injury, a significant decrease compared to pre-injury baseline in the force threshold required to elicit a withdrawal response was evident both ipsilaterally (E) and contralaterally (F) for each of the six weeks following injury. The decrease in ipsilateral forepaw withdrawal latency and bilateral force threshold in injured animals occurred in the absence of changes in grip strength in either forepaw (G–H). Epi = epicenter; IB = immunoblotting; histo = histology; * = p<0.05; ** = p<0.01; **** = p<0.0001.</p
<p>Astrocyte activation in the superficial dorsal horn was characterized by quantification ... more <p>Astrocyte activation in the superficial dorsal horn was characterized by quantification of GFAP expression in laminae I–II. (A) and (B) show representative images of dorsal horn GFAP expression at high magnification in laminectomy and injured animals, respectively. At the injury epicenter, compared to uninjured control animals (C), an increase in GFAP expression in the ipsilateral dorsal horn was evident at two (D) and six weeks (E) following injury. No significant changes in GFAP expression were observed at the other regions studied (F). IF = immunofluorescence; * = p<0.05; *** = p<0.001.</p
<p>Immunohistochemical analysis revealed a decrease in GLT1 protein expression two (B) and ... more <p>Immunohistochemical analysis revealed a decrease in GLT1 protein expression two (B) and six weeks (C) after cervical contusion SCI compared to laminectomy (A). This downregulation of the glutamate transporter was seen in the superficial dorsal horn on both sides of the spinal cord both at the injury site and caudal to the injury (D). Immunoblots of spinal cord tissue also showed a significant loss of GLT1 expression on the ipsilateral side at both time points following injury. However, this difference was seen only at the epicenter (E) and not caudal to the injury (F). Representative immunoblots are shown for each region (E–F). Analysis of GLT1 levels in regions of the spinal cord other than the superficial laminae at six weeks after injury reveal a loss of GLT1 expression in the ventral horn at the epicenter with no changes caudal to the injury (G). IF = immunofluorescence; epi = epicenter; ipsi = ipsilateral; contra = contralateral; lam = laminectomy; * = p<0.05; ** = p<0.01; *** = p 0.001.</p
<p>In the superficial laminae of the injured ipsilateral dorsal horn, we observed cells co-... more <p>In the superficial laminae of the injured ipsilateral dorsal horn, we observed cells co-expressing GFAP and Ki67 (A), representing activated and proliferating astrocytes. In laminectomy control animals, little to no cell proliferation was evident (B). However, the number of Ki67-positive cells was significantly increased at two weeks (C) and six weeks (D) after injury on the ipsilateral side both at the level of and caudal to the injury (E). Additionally, at two weeks, there was a significant increase in proliferating cells contralaterally at the injury site (E). At both two and six weeks, a significant percentage of Ki67-positive cells were also either GFAP-positive or CD11b-positive (F). Compared to laminectomy (G), the intensity of CD11b expression in the superficial dorsal horn was greater in animals with C6 SCI at both two (H–I) and six (I) weeks after injury. This increase was significant at the injury site both ipsilaterally and contralaterally, as well as caudal to the injury on the contralateral side (I). * = p<0.05; ** = p<0.01; **** = p<0.0001.</p
<p>Harvested tissue was stained with Cresyl violet and Eriochrome cyanine. At the level of ... more <p>Harvested tissue was stained with Cresyl violet and Eriochrome cyanine. At the level of the laminectomy, uninjured control animals (A) exhibited large motor neurons in the ventral horn (arrowheads). At six weeks post-injury, animals receiving unilateral C5 (B) or C6 (C) contusion SCI showed a loss of these motor neurons at the injury epicenter but not 1.0 mm caudal to the injury (D). The spread of ventral horn motor neuron loss was approximately 1.0 mm rostrally and 1.0 mm caudally (2.0 mm total) from the epicenter (E). Immunohistochemical analyses of the injury models were performed in laminae I–II and lamina III of the cervical spinal cord dorsal horn (F–G). Lam = laminectomy.</p
<p>Following cervical contusion SCI, GLT1 expression/promoter activity, represented by eGFP... more <p>Following cervical contusion SCI, GLT1 expression/promoter activity, represented by eGFP-positive cells, was decreased in the superficial dorsal horn. In laminectomy animals, there were low levels of GFAP, although many of the cells that were GFAP-positive also expressed GLT1 (A–B). Following injury, however, an increase in GFAP was observed, but fewer of these activated astrocytes co-expressed GLT1 (C). Laminectomy control animals (D) had greater numbers of eGFP-positive cells compared to animals that received contusion injury two days, two weeks, or six weeks (E) prior to analysis. This decrease in GLT1 expression was observed at the injury epicenter in both the ipsilateral (F) and contralateral (G) superficial laminae as well as caudal to the injury on the ipsilateral side (H). No change in the number of eGFP-positive cells was found caudal to the injury on the contralateral side (I). * = p<0.05; ** = p<0.01; *** = p 0.001.</p
<p>ΔfosB staining was used to measure the extent of persistent neuronal activation in the s... more <p>ΔfosB staining was used to measure the extent of persistent neuronal activation in the spinal cord. Following injury, ΔfosB-positive nuclei (arrowheads) were evident in laminae I–II (A–C). At all regions, laminectomy control animals (A) showed little-to-no ΔfosB expression (D). Two weeks after C6 injury (B), a significant increase in numbers of ΔfosB expressing cells in the superficial laminae of the ipsilateral dorsal horn was observed at the injury epicenter and caudal to the injury (D). ΔfosB levels were further increased at all regions in animals sacrificed six weeks post-injury (C, D). * = p<0.05; ** = p<0.01; *** = p 0.001; **** = p<0.0001.</p
Development of neuropathic pain occurs in a major portion of traumatic spinal cord injury (SCI) p... more Development of neuropathic pain occurs in a major portion of traumatic spinal cord injury (SCI) patients, resulting in debilitating and often long-term physical and psychological burdens. Following SCI, chronic dysregulation of extracellular glutamate homeostasis has been shown to play a key role in persistent central hyperexcitability of superficial dorsal horn neurons that mediate pain neurotransmission, leading to various forms of neuropathic pain. Astrocytes express the major CNS glutamate transporter, GLT1, which is responsible for the vast majority of functional glutamate uptake, particularly in the spinal cord. In our unilateral cervical contusion model of mouse SCI that is associated with ipsilateral forepaw heat hypersensititvity (a form of chronic at-level neuropathic pain-related behavior), we previously reported significant and long-lasting reductions in GLT1 expression and functional GLT1-mediated glutamate uptake in cervical spinal cord dorsal horn. To therapeutically ...
Transplantation-based replacement of lost and/or dysfunctional astrocytes is a promising therapy ... more Transplantation-based replacement of lost and/or dysfunctional astrocytes is a promising therapy for spinal cord injury (SCI) that has not been extensively explored, despite the integral roles played by astrocytes in the central nervous system (CNS). Induced pluripotent stem (iPS) cells are a clinically-relevant source of pluripotent cells that both avoid ethical issues of embryonic stem cells and allow for homogeneous derivation of mature cell types in large quantities, potentially in an autologous fashion. Despite their promise, the iPS cell field is in its infancy with respect to evaluating in vivo graft integration and therapeutic efficacy in SCI models. Astrocytes express the major glutamate transporter, GLT1, which is responsible for the vast majority of glutamate uptake in spinal cord. Following SCI, compromised GLT1 expression/function can increase susceptibility to excitotoxicity. We therefore evaluated intraspinal transplantation of human iPS cell-derived astrocytes (hIPSA...
Amyotrophic lateral sclerosis (ALS) is characterized by relatively rapid degeneration of both upp... more Amyotrophic lateral sclerosis (ALS) is characterized by relatively rapid degeneration of both upper and lower motor neurons, with death normally occurring 2-5years following diagnosis primarily due to respiratory paralysis resulting from phrenic motor neuron (PhMN) loss and consequent diaphragm denervation. In ALS, cellular abnormalities are not limited to MNs. For example, decreased levels and aberrant functioning of the major central nervous system (CNS) glutamate transporter, GLT1, occur in spinal cord and motor cortex astrocytes of both humans with ALS and in SOD1(G93A) rodents, a widely studied ALS animal model. This results in dysregulation of extracellular glutamate homeostasis and consequent glutamate excitotoxicity, a primary mechanism responsible for MN loss in ALS animal models and in the human disease. Given these observations of GLT1 dysfunction in areas of MN loss, as well as the importance of testing therapeutic strategies for preserving PhMNs in ALS, we evaluated int...
The Journal of neuroscience : the official journal of the Society for Neuroscience, Jan 21, 2015
Spinal cord injury (SCI) patients develop chronic pain involving poorly understood central and pe... more Spinal cord injury (SCI) patients develop chronic pain involving poorly understood central and peripheral mechanisms. Because dysregulation of the voltage-gated Kv3.4 channel has been implicated in the hyperexcitable state of dorsal root ganglion (DRG) neurons following direct injury of sensory nerves, we asked whether such a dysregulation also plays a role in SCI. Kv3.4 channels are expressed in DRG neurons, where they help regulate action potential (AP) repolarization in a manner that depends on the modulation of inactivation by protein kinase C (PKC)-dependent phosphorylation of the channel's inactivation domain. Here, we report that, 2 weeks after cervical hemicontusion SCI, injured rats exhibit contralateral hypersensitivity to stimuli accompanied by accentuated repetitive spiking in putative DRG nociceptors. Also in these neurons at 1 week after laminectomy and SCI, Kv3.4 channel inactivation is impaired compared with naive nonsurgical controls. At 2-6 weeks after laminect...
The Journal of neuroscience : the official journal of the Society for Neuroscience, Jan 28, 2014
A major portion of spinal cord injury (SCI) cases affect midcervical levels, the location of the ... more A major portion of spinal cord injury (SCI) cases affect midcervical levels, the location of the phrenic motor neuron (PhMN) pool that innervates the diaphragm. While initial trauma is uncontrollable, a valuable opportunity exists in the hours to days following SCI for preventing PhMN loss and consequent respiratory dysfunction that occurs during secondary degeneration. One of the primary causes of secondary injury is excitotoxic cell death due to dysregulation of extracellular glutamate homeostasis. GLT1, mainly expressed by astrocytes, is responsible for the vast majority of functional uptake of extracellular glutamate in the CNS, particularly in spinal cord. We found that, in bacterial artificial chromosome-GLT1-enhanced green fluorescent protein reporter mice following unilateral midcervical (C4) contusion SCI, numbers of GLT1-expressing astrocytes in ventral horn and total intraspinal GLT1 protein expression were reduced soon after injury and the decrease persisted for ≥6 weeks...
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