pubmed.ncbi.nlm.nih.gov

TrkB.T1 contributes to neuropathic pain after spinal cord injury through regulation of cell cycle pathways - PubMed

  • ️Tue Jan 01 2013

TrkB.T1 contributes to neuropathic pain after spinal cord injury through regulation of cell cycle pathways

Junfang Wu et al. J Neurosci. 2013.

Abstract

Spinal cord injury (SCI) frequently causes severe, persistent central neuropathic pain that responds poorly to conventional pain treatments. Brain-derived neurotrophic factor (BDNF) signaling appears to contribute to central sensitization and nocifensive behaviors in certain animal models of chronic pain through effects mediated in part by the alternatively spliced truncated isoform of the BDNF receptor tropomyosin-related kinase B.T1 (trkB.T1). Mechanisms linking trkB.T1 to SCI-induced chronic central pain are unknown. Here, we examined the role of trkB.T1 in central neuropathic pain after spinal cord contusion. Genetic deletion of trkB.T1 in mice significantly reduced post-SCI mechanical hyperesthesia, locomotor dysfunction, lesion volumes, and white matter loss. Whole genome analysis, confirmed at the protein level, revealed that cell cycle genes were upregulated in trkB.T1(+/+) but not trkB.T1(-/-) spinal cord after SCI. TGFβ-induced reactive astrocytes from WT mice showed increased cell cycle protein expression that was significantly reduced in astrocytes from trkB.T1(-/-) mice that express neither full-length trkB nor trkB.T1. Administration of CR8, which selectively inhibits cyclin-dependent kinases, reduced hyperesthesia, locomotor deficits, and dorsal horn (SDH) glial changes after SCI, similar to trkB.T1 deletion, without altering trkB.T1 protein expression. In trkB.T1(-/-) mice, CR8 had no effect. These data indicate that trkB.T1 contributes to the pathobiology of SCI and SCI pain through modulation of cell cycle pathways and suggest new therapeutic targets.

PubMed Disclaimer

Figures

Figure 1.
Figure 1.

The trkB.T1 protein is significantly upregulated at the thoracic injured area and lumbar spinal cord horn after SCI. A, Schematic drawing of the area of injury and region of tissue harvested. A moderate contusion injury was produced using a spinal cord impactor with a force of 60 kdyn. Five millimeters of spinal cord tissue centered on the epicenter or equivalent tissue from a laminectomy control was processed for Western blot and microarray analysis. B–C, Representative Western blot showing trkB.FL and trkB.T1 in the intact spinal cord (control), at 1 d and 8 weeks after SCI (D1 and WK8 SCI) in trkB.T1+/+ and trkB.T1−/− mice. D–E, In the trkB.T1+/+ mice, trkB.T1 protein expression was significantly upregulated 1 d after SCI (n = 4) compared with control (n = 4). The upregulation was sustained at week 8 (n = 8 per group, p < 0.05 SCI vs control, two-tailed Student's t test). There was no difference in the expression of the trkB.FL protein at 1 d after SCI or at week 8 after SCI compared with control (n = 4 per group). F, Representative Western blot showing trkB.T1 expression at the lumbar SDH in the intact spinal cord (control) at 1 and 3 d after SCI (D1 and D3 SCI) in trkB.T1+/+ and trkB.T1−/− mice. G, TrkB.T1 expression in TrkB.T1+/+ mice was significantly upregulated at 24 h after thoracic injury (p < 0.05, D1 SCI vs control) and remained elevated at 3 d (n = 4 mice per group). Data are expressed as mean ± SEM.

Figure 2.
Figure 2.

Gene deletion of trkB.T1 results in improved locomotor functional recovery and decreased mechanical hyperesthesia after SCI. A, BMS scores in trkB.T1−/− mice (♦; n = 15) were significantly higher than in trkB.T1+/+ mice (◊; n = 15) starting at day 14 after SCI (p < 0.05, two-way ANOVA with repeated measures). B, TrkB.T1−/− mice (♦; n = 12) had significantly less post-SCI mechanical hyperesthesia than trkB.T1+/+ mice (◊; n = 12, p < 0.05, Mann–Whitney U test). C, Hindpaw mechanical withdrawal stimulus-response curves in intact naive mice and after SCI demonstrate that, after injury, the trkB.T1+/+ mice (◊; n = 12) withdrew their paws from the mechanical stimuli more frequently than the trkB.T1−/− mice (♦; n = 12, p < 0.05, one-tailed Student's t test). D, A series of representative ECRC-stained tissue sections from trkB.T1+/+ and trkB.T1−/− mice at the epicenter (Epi) and rostral (R1–R4) and caudal (C1–C4) to the injury. ECRC stains myelinated areas of spared WM. E, Quantification of the total WM area (mm2) in stained tissue sections showed a significant increase in the WM area of trkB.T1−/− mice (n = 7) compared with trkB.T1+/+ (n = 7, p < 0.05, one-tailed Student's t test vs trkB.T1+/+ mice). Data are expressed as mean ± SEM.

Figure 3.
Figure 3.

TrkB.T1 KO reduces secondary injury after SCI. A, Representative histologically stained tissue sections 1 mm rostral to the epicenter from a sham trkB.T1+/+ mouse and from injured trkB.T1+/+ and trkB.T1−/− mice. The SCI lesion is the region with less GFAP/DAB staining than surrounding tissue. B, The lesion volumes of trkB.T1−/− mice (n = 8) were significantly smaller than in trkB.T1+/+ mice (n = 8) at 8 weeks after SCI (*p < 0.05, one-tailed Student's t test). C–D, Western blot analysis showed that gene deletion of trkB.T1 (n = 8) significantly reduced the SCI-induced upregulation of Iba-1 protein at 8 weeks after injury compared with trkB.T1+/+ mice (n = 8). Five-millimeter segments of spinal cord tissue representing the injury epicenter were collected for Western blot analysis. Representative immunoblots are shown in C. E–F, Western blot analysis showed that deleting trkB.T1 (n = 8) significantly reduced SCI-induced upregulation of GFAP, CHL1, and NG2 at 8 weeks after injury compared with trkB.T1+/+ mice (n = 8). Five-millimeter segments of spinal cord tissue representing the injury epicenter were collected for Western blot analysis. Representative immunoblots are shown in E. The data in D, F were analyzed by one-way ANOVA with post hoc analysis using Student–Newman–Keuls test. *p < 0.05, WT SCI versus WT control; #p < 0.05, KO SCI versus WT SCI. Data are expressed as mean ± SEM.

Figure 4.
Figure 4.

TrkB.T1 KO results in decreased gliopathy at the lumbar SDH after SCI. A–C, Western blot analysis showed that GFAP and Iba-1 protein expression at the lumbar SDH were elevated at 1 d after injury in the trkB.T1+/+ mice and were significantly higher at day 3 in the trkB.T1+/+ mice than that in the trkB.T1−/− mice. There was no difference in the expression levels for GFAP and Iba-1 between genotypes in the uninjured control state. n = 4 mice per group; *p < 0.05, WT D1/D3 SCI versus WT control; #p < 0.05, KO D1 SCI versus WT D1 SCI; and $p < 0.05, KO D3 SCI versus WT D3 SCI. D–F, Quantification of ED1+ (green)/Iba-1+ (red) activated microglial cells showed that after SCI, there were significantly more activated microglia in the lumbar SDH of trkB.T1+/+ mice (n = 7) compared with trkB.T1−/− mice (n = 7). The number of GFAP-positive cells (green) with a swollen hypertrophic appearance was significantly increased at 7 d after injury in both trkB.T1+/+ (n = 7) and trkB.T1−/− mice (n = 7). Deleting trkB.T1 significantly reduced the number of reactive astrocytes in the trkB.T1−/− mice compared with WT. Scale bar, 250 μm for top and 100 μm for bottom in D. The data in B, C and E, F were analyzed by one-way ANOVA with post hoc analysis using a Student–Newman–Keuls test. *p < 0.05, WT SCI versus WT control; #p < 0.05, KO SCI versus WT SCI. Data are expressed as mean ± SEM.

Figure 5.
Figure 5.

Cell cycle genes are upregulated in the spinal cord of trkB.T1+/+ mice early after SCI but not in trkB.T1-null mice. A, Differential gene expression analysis in the spinal cord over time comparing differentially expressed genes in the trkB.T1−/− mice compared with WT mice demonstrates that the largest number of genes is altered early at day 1 after SCI. The Venn diagrams show each time point (days 1, 3, and 7 after SCI), with the number of differentially expressed genes at each. B, Heat map of genes that cluster by genotype at day 1 in the trkB.T1−/− spinal cord compared with WT. The normalized expression of the summarized probe sets is shown. The histogram key indicates that green genes are downregulated and red genes are upregulated. C, Unbiased pathway analysis demonstrates significant downregulation of cell cycle pathways in trkB.T1−/− spinal cord compared with WT at day 1. The top 10 differentially enriched canonical signaling pathways are shown. Green indicates downregulation and red indicates upregulation. The orange line shows the −log10 p for each pathway. D, Overlapping, shared gene set analysis was performed to determine whether there are shared genes across a number of significantly regulated canonical signaling pathways. Each pathway is displayed as a single node. The darker the red, the more significant the overlapping pathway is in our gene set by the Fisher's exact test p-value. This diagram depicts the top 25 significantly regulated pathways in which our gene set has shared genes across pathways.

Figure 6.
Figure 6.

Deleting trkB.T1 attenuates upregulation of cell-cycle-related proteins in the lesioned and lumbar areas of post-SCI spinal cord and reactive astrocytes in vitro. A, Cdk1 is significantly upregulated 24 h after SCI in trkB.T1+/+ spinal cord by microarray (n = 4, 5.9 FC, FDR p = 2.09E-10) and qPCR (n = 4, FC 4.16, p = 0.0022), but not in trkB.T1−/− mice (n = 4). B, Representative immunoblots of E2F1, CDK1, and cyclin E in spinal cord tissue from trkB.T1+/+ and trkB.T1−/− mice 24 h after SCI. C, Quantification showing E2F1, CDK1, and cyclin E protein upregulation in trkB.T1+/+ and trkB.T1−/− mice compared with controls, which was significantly attenuated in trkB.T1−/− compared with trkB.T1+/+ mice (n = 4 mice/group). D, Representative immunoblots of cyclin D1, CDK4, and PCNA in spinal cord tissue from trkB.T1+/+ and trkB.T1−/− mice 8 weeks after SCI. E, Quantification showing upregulation of all three proteins in trkB.T1+/+ and CDK4 and PCNA in trkB.T1−/− mice compared with the controls, which was significantly attenuated in trkB.T1−/− compared with trkB.T1+/+ mice (n = 8 mice/group). *p < 0.05, WT SCI versus WT control; #p < 0.05, KO SCI versus WT SCI. F, Representative immunoblots of PCNA and CDK4 in primary astrocyte cultures from trkB.T1+/+ and trkB.T1−/− mice ± TGFβ1. G, Quantification showed significant attenuation of protein upregulation in reactive astrocytes from trkB.T1−/− compared with trkB.T1+/+ mice. *p < 0.05, WT TGFβ1 versus WT control; #p < 0.05, KO TGFβ1 versus WT-TGFβ1; n = 4 independent cultures/group.

Figure 7.
Figure 7.

Deleting trkB.T1 attenuates upregulation of cell-cycle-related proteins in the lumbar SDH after SCI. A–D, Western blot analysis showing that 1 d after SCI, expression levels of E2F1, CDK1, and PCNA in the lumbar SDH were markedly increased in the trkB.T1+/+ mice, but significantly lower in trkB.T1−/− mice. Elevation of E2F1 was transit only at 24 h after SCI, but at 3 d after injury, the expression of CDK1 and PCNA remained higher in the trkB.T1+/+ mice than that in the trkB.T1−/− mice (n = 4 mice per group). *p < 0.05, WT D1/D3 SCI versus WT control; #p < 0.05, KO D1 SCI versus WT D1 SCI; and $p < 0.05, KO D3 SCI versus WT D3 SCI. E, Representative coronal sections of lumbar spinal cord stained for PCNA and CDK4. F, Few PCNA+ cells were found in the SDH of control mice. PCNA+ cells increased in trkB.T1+/+ (n = 6) and trkB.T1−/− mice (n = 6) 1 d after SCI compared with controls (n = 4), which was significantly attenuated in trkB.T1−/− compared with trkB.T1+/+ mice. G, Few CDK4+ cells were found in the SDH of control mice. CDK4+ cells increased in trkB.T1+/+ (n = 7) and trkB.T1−/− mice (n = 7) 8 weeks after SCI compared with controls (n = 4), which was significantly attenuated in trkB.T1−/− compared with trkB.T1+/+ mice. Scale bar, 100 μm. The data in B–D and F–G were analyzed by one-way ANOVA with post hoc analysis using Student–Newman–Keuls test. *p < 0.05, WT SCI versus WT control; #p < 0.05, KO SCI versus WT SCI. Data are expressed as mean ± SEM.

Figure 8.
Figure 8.

The CDK inhibitor CR8 decreased mechanical hyperesthesia and improved locomotor function in trkB.T1+/+ but not trkB.T1−/− mice. Intrathecal CR8 (1 m

m

/5 μl) or saline was given immediately after SCI and for 6 subsequent days. Assessments occurred before and on days 1, 3, 7, 14, 21, 28, 35, and 42 post-SCI for locomotor function and on days 28, 35, and 42 post-SCI for mechanical sensitivity. A, CR8-treated trkB.T1+/+ mice (n = 19) had significantly higher BMS scores on days 14–42 compared with saline-treated mice (n = 19; p < 0.05, two-way ANOVA with repeated measures). B, CR8-treated trkB.T1+/+ mice (n = 10) had significantly higher mechanical thresholds on days 28–42 than saline-treated mice (n = 10; Mann–Whitney U test, p < 0.05). There was no effect of CR8 on sham-SCI mice (n = 6/group). C–F, Stimulus-response curves in intact naive mice and at 28, 35, and 42 d after SCI demonstrate that the saline-treated SCI mice (n = 10) withdrew their paws from the mechanical stimuli significantly more frequently than the CR8-treated SCI mice (n = 10; one-way ANOVA; *p < 0.05). There was no difference between the stimulus-response curves of the CR8-treated sham and saline-treated sham mice.

Figure 9.
Figure 9.

Cell cycle inhibition by CR8 has no effects on behavior and cell cycle activation in the trkB.T1−/− mice after SCI. A, CDK4, PCNA, and E2F5 expression were upregulated 8 weeks after injury in trkB.T1+/+ mice. B, CR8 treatment (n = 6) significantly attenuated SCI-induced protein upregulation compared with saline (n = 5; n = 3 control, n = 5 saline, n = 6 CR8). *p < 0.05, vehicle versus control; #p < 0.05, CR8 versus vehicle. C, Representative immunoblot showing increased trkB.T1 expression 8 weeks after SCI, with trkB.FL remaining unchanged. D, There was no difference in trkB.T1 expression between CR8-treated mice (n = 5) and saline-treated trkB.T1+/+ mice (n = 3). E, There was no difference in locomotor function between CR8-treated (n = 13) and saline-treated (n = 15) trkB.T1−/− mice after SCI. F, There was no difference in mechanical hyperesthesia between CR8-treated (n = 11) and saline-treated (n = 11) trkB.T1−/− mice after SCI. There was no CR8 effect on sham-SCI mice (n = 6/group). G–H, CDK4, PCNA, and E2F5 expression did not differ between CR8-treated (n = 6) and saline-treated (n = 5) trkB.T1−/− mice 8 weeks after SCI. Data are expressed as mean ± SEM.

Similar articles

Cited by

References

    1. Armanini MP, McMahon SB, Sutherland J, Shelton DL, Phillips HS. Truncated and catalytic isoforms of trkB are co-expressed in neurons of rat and mouse. Eur J Neurosci. 1995;7:1403–1409. doi: 10.1111/j.1460-9568.1995.tb01132.x. - DOI - PubMed
    1. Basso DM, Fisher LC, Anderson AJ, Jakeman LB, McTigue DM, Popovich PG. Basso Mouse Scale for locomotion detects differences in recovery after spinal cord injury in five common mouse strains. J Neurotrauma. 2006;23:635–659. doi: 10.1089/neu.2006.23.635. - DOI - PubMed
    1. Baxter GT, Radeke MJ, Kuo RC, Makrides V, Hinkle B, Hoang R, Medina-Selby A, Coit D, Valenzuela P, Feinstein SC. Signal transduction mediated by the truncated trkB receptor isoforms, trkB.T1 and trkB.T2. J Neurosci. 1997;17:2683–2690. - PMC - PubMed
    1. Bush TG, Puvanachandra N, Horner CH, Polito A, Ostenfeld T, Svendsen CN, Mucke L, Johnson MH, Sofroniew MV. Leukocyte infiltration, neuronal degeneration, and neurite outgrowth after ablation of scar-forming, reactive astrocytes in adult transgenic mice. Neuron. 1999;23:297–308. doi: 10.1016/S0896-6273(00)80781-3. - DOI - PubMed
    1. Byrnes KR, Stoica BA, Fricke S, Di Giovanni S, Faden AI. Cell cycle activation contributes to post-mitotic cell death and secondary damage after spinal cord injury. Brain. 2007;130:2977–2992. doi: 10.1093/brain/awm179. - DOI - PubMed

Publication types

MeSH terms

Substances