Figure 1.
Animals receiving unilateral cervical contusion SCI exhibited persistent thermal hyperalgesia and tactile allodynia in the forepaw.
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.
Figure 2.
Unilateral contusion SCI induced a loss of ventral horn motor neurons at the injury epicenter.
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.
Figure 3.
Chronic neuronal activation in the dorsal horn resulted from cervical contusion SCI.
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.
Figure 4.
Astrocytes were activated and proliferated in the dorsal horn following cervical contusion SCI.
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.
Figure 5.
Enhanced cell proliferation, including proliferation of astrocytes, was evident after cervical contusion SCI.
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.
Figure 6.
Astrocyte GLT1 promoter activity was reduced in injured BAC-GLT1-eGFP transgenic reporter mice.
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.
Figure 7.
Cervical contusion SCI resulted in decreased astrocyte GLT1 expression in the dorsal horn.
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.