Soluble TNFα Signaling within the Spinal Cord Contributes to the Development of Autonomic Dysreflexia and Ensuing Vascular and Immune Dysfunction after Spinal Cord Injury

Intrathecal administration of XPro1595 diminishes signaling downstream of persistent sTNFα/TNFR1 expression below a high-thoracic SCI. a, Levels of TNFα protein in lumbar spinal cord were assessed via ELISA in naive rats and injured rats at different after T3Tx time points (3 d, 4 weeks; n = 4 or 5 per group). Even 1 month after injury, sTNFα levels well below a T3Tx were significantly higher than in naive lumbar spinal cord. b–e, An expression time course of TNFR1 or the phosphorylated (i.e. activated) p65 subunit of NF-κB (pNF-κB p65), a downstream effector of TNFR1 activation, in lumbar spinal cord of uninjured rats or T3Tx rats treated with saline, was determined via Western blot (n = 3 per group). Levels of TNFR1 (b, c) and pNF-κB p65 (d, e) well below a T3Tx were significantly higher than that observed in naive controls, regardless of time point, after SCI. Additionally, there was a significant spike in both TNFR1 and pNF-kB p65 levels between 2 and 3 weeks after T3Tx. f–i, Expression levels of TNFR1 and pNF-κB p65 in lumbar spinal cord of naive rats or T3Tx rats treated with saline or the sTNFα biologic XPro1595 4 weeks after SCI were determined via Western blot (n = 4 or 5 per group). Levels of TNFR1 well below a T3Tx were significantly higher than that observed in naive controls, regardless of whether saline or XPro1595 was infused (f, g). However, despite similarities in TNFR1 expression, inhibiting sTNFα signaling with XPro1595 significantly diminished levels of pNF-κB p65, compared with T3Tx-saline animals. Levels in T3Tx-XPro1595 animals were indistinguishable from that observed in naive animals (h, i). Data are mean ± SEM. p values were determined by a Student's t test or one-way ANOVA and post hoc Fisher's LSD tests: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ^#p < 0.0001 versus 2 weeks. ^%p < 0.001 versus 2 weeks. ^@p < 0.01.

Inhibiting sTNFα/TNFR1 dampens microglial reactivity but does not reduce astrocytic gliosis. a–c, Representative confocal images of immunostaining of Iba-1⁺ microglia in lumbar spinal cord from naive, uninjured animals or T3Tx animals 4 weeks after SCI that were treated with either saline or XPro1595 (n = 3 per group). In naive animals, Iba-1⁺ microglia took on a highly ramified morphology typical of resting microglia (a, a′). There was significantly more Iba-1⁺ immunoreactivity in tissue from T3Tx-saline animals (d). The Iba-1⁺ microglia in these animals had short processes and took on the phenotype of more reactive cells (b, b′). Iba-1⁺ immunoreactivity in T3Tx-XPro1595 animals were less than their saline-treated counterparts but also were greater than naive levels (c, c′, d). e–g, Representative confocal images of immunostaining of GFAP⁺ astrocytes in lumbar spinal cord from naive, uninjured animals or T3Tx animals 4 weeks after SCI that were treated with either saline or XPro1595 (n = 4 per group). Higher magnification images of the boxed regions in e–g are shown in e′–g′. Astrocytes in naive animals show morphology typical of a resting astrocyte (e). T3Tx resulted in significantly more GFAP⁺ immunoreactivity, regardless of treatment (f–h). The GFAP⁺ astrocytes in both T3Tx-saline and -XPro1595 animals appeared hypertrophic and took on a phenotype typical of more reactive astrocytes (f, g). There was no difference in GFAP⁺ immunoreactivity between treatment groups (h). Data are mean ± SEM. *p < 0.05 (one-way ANOVA and post hoc Fisher's LSD test). **p < 0.01 (one-way ANOVA and post hoc Fisher's LSD test). ****p < 0.0001 (one-way ANOVA and post hoc Fisher's LSD test). Scale bars: a–c, 37.5 μm; e–g, 25 μm.

XPro1595 does not affect basal hemodynamics. T3Tx animals treated with XPro1595 or saline (n = 8 or 9 per group) were implanted with radiotelemetry pressure transducers into the descending aorta to allow for recording BP and HR in animals at various time points. Baseline MAP (a) and HR (b) of T3Tx-saline and T3Tx-XPro1595 were similar at all time points, indicating that XPro1595 did not grossly affect hemodynamics.

XPro1595 treatment mitigates colorectal distension-induced AD. At 2, 3, and 4 weeks after T3Tx, HR and BP were determined in rats before, during, and after CRD, a well-established experimental means to elicit an AD episode that mimics constipation, a common trigger in SCI patients. a, b, Representative beat-to-beat arterial pressure traces from T3Tx-saline and T3Tx-XPro1595 animals 4 weeks after SCI. Red line indicates the 1 min CRD interval. White line indicates MAP. T3Tx-saline animals had stereotypical sharp spikes in MAP in response to CRD. T3Tx-XPro1595 animals exhibited a dramatically smaller increase in MAP. c, Quantification of the increase in MAP above baseline in response to CRD. The MAP increase in XPro1595-treated animals was significantly smaller than in saline-treated animals at every time point. Furthermore, there was a strong trend that the BP spike in the saline-treated animals progressively increased from 2 to 4 weeks (p = 0.054), further suggesting that AD worsens over time. Conversely, the MAP increase in the T3Tx-XPro1595 animals was stable at all 3 time points. d, The time that it took to return to baseline MAP after 1 min of CRD was significantly longer in T3Tx-saline animals than T3Tx-XPro1595 animals. e, We observed no significant differences in bradycardia (bpm; beats per minute) in response to CRD between groups, although there was a strong trend at 4 weeks (p = 0.051). Data are mean ± SEM. *p < 0.05 (two-way ANOVA and post hoc Fisher's LSD test). **p < 0.01 (two-way ANOVA and post hoc Fisher's LSD test). ***p < 0.001 (two-way ANOVA and post hoc Fisher's LSD test). ****p < 0.0001 (two-way ANOVA and post hoc Fisher's LSD test).

Inhibiting sTNFα signaling decreases the frequency and severity of naturally occurring AD episodes after complete T3 SCI. At 2, 3, and 4 weeks after injury, BP and HR were continuously recorded over 24 h in T3Tx animals treated with XPro1595 or saline (n = 8–9 per group) while they moved freely in their cages. a, b, Representative MAP traces from a T3Tx-saline or a T3Tx-XPro1595 animal over a 24 h period 4 weeks after injury. Red asterisks indicate naturally occurring (i.e., not experimentally induced) AD events detected using MATLAB followed by manual verification. c, Quantification of the frequency of naturally occurring events at 2, 3, and 4 weeks after T3Tx. At 2 weeks, animals from both groups experienced similar numbers of AD episodes. The number of events did not change over time in the XPro1595-treated animals. However, the number of daily events the saline-treated animals experienced significantly increased by 4 weeks. Furthermore, these animals had sharply more episodes than their XPro1595-treated counterparts. d, T3Tx-XPro1595 animals had significantly lower peak MAP during each detected event at all time points assessed. Furthermore, while the highest MAP in the XPro1595-treated animals across weeks were similar, the peak MAP in the saline-treated animals significantly increased over time. e, The time that MAP remained >20 mmHg above baseline was considered the duration of each naturally occurring AD event. XPro1595-treated T3Tx rats had significantly shorter events than saline-treated rats at 3 weeks. Data are mean ± SEM. *p < 0.05 (two-way ANOVA and post hoc Fisher's LSD test). ****p < 0.0001 (two-way ANOVA and post hoc Fisher's LSD test).

Intrathecally administered XPro1595 diminishes peripheral vascular dysfunction after high-level SCI. Mesenteric arteries were harvested from naive animals (n = 5) or T3Tx-saline (n = 7) and T3Tx-XPro1595 rats (n = 6) 4 weeks after SCI for ex vivo analysis. a, b, The lumen diameters and wall thicknesses of isolated small mesenteric arteries were determined. Arteries from T3Tx-saline animals had a significantly narrower lumen and a thinner wall than those from naive animals. Arteries from T3Tx-XPro1595 animals were not statistically different from naive animals. c–e, We assessed mesenteric artery reactivity to increasing concentrations of the vasopressor PE. There was a leftward shift of the PE concentration-response curve for arteries from T3Tx-saline animals (c). Moreover, the concentration of PE required for arteries to reach 50% constriction (pEC50) was lowest in T3Tx-saline animals (which experienced the most severe AD) (d), indicating that arteries from T3Tx-saline animals were hyper-responsive to PE. XPro1595 decreased percentage maximum constriction in response to PE (e). f, g, We also examined the response of the isolated arteries to the endothelium-dependent vasodilator ACh. We did not observe any group differences in ACh-induced vasodilation at any concentration. The concentration-response curves (f), pEC50 (g), and percentage maximal dilation (h) of the arteries from the four groups were similar, indicating that the enhanced constriction (c, d) was not due to diminished vasodilation. Data are mean ± SEM. p values were determined by one-way ANOVA and post hoc Fisher's LSD tests: ^∧T3Tx-saline versus Naive (p < 0.01); T3Tx-saline versus T3Tx-XPro1595 (p < 0.05). ^#T3Tx-saline versus T3Tx-XPro1595 (p < 0.05). *p < 0.05.

Intrathecally administering XPro1595 averts peripheral immune dysfunction after T3 SCI. Spleens from naive, uninjured animals and T3Tx-saline and -XPro1595 animals 4 weeks after SCI were harvested and processed for flow cytometry (n = 6 per group). a, b, T3Tx-saline animals had a smaller proportion of total leukocytes that were CD45R⁺ B cells (a) or CD8⁺ T cells (b) than naive and T3Tx-XPro1595 animals. There was no significant difference in the percentage of cells containing these markers between naive and T3Tx-XPro1595 animals. c, The proportion of leukocytes that were CD4⁺ T cells was similar across groups. d, T3Tx-XPro1595 animals had a greater percentage of CD4⁺ T cells that were also CD25⁺ FoxP3⁺ regulatory T cells (T_Reg) than both uninjured and T3Tx-saline animals. e, T3Tx-saline animals had a higher percentage of total leukocytes that were CD11b/c⁺ than both uninjured and T3Tx-XPro1595 animals. f, Moreover, T3Tx-saline animals had a greater percentage that were CD68⁺ monocytes/macrophages than uninjured animals. T3Tx-XPro1595 animals had levels of CD68⁺ monocytes/macrophages that were in between and statistically similar to those seen in naive and T3Tx-saline animals. Data are mean ± SEM. *p < 0.05 (one-way ANOVA and post hoc Fisher's LSD test). **p < 0.01 (one-way ANOVA and post hoc Fisher's LSD test). ****p < 0.0001 (one-way ANOVA and post hoc Fisher's LSD test).

Inhibiting sTNFα signaling decreases CRD-elicited activation of interneurons caudal to T3 SCI. a–i, Activated c-Fos⁺ (green) interneurons after CRD were immunohistochemically assessed in T3Tx-saline or T3Tx-XPro1595 animals 4 weeks after SCI (n = 3 or 4/group). CRD in T3Tx-saline animals induced c-Fos in many interneurons within L6 dorsal horn (a, c) and dorsal gray commissure in laminae VII/X (d, f), which contains neurons that relay visceral sensory input (e.g., CRD) to SPNs in thoracic cord. There were fewer c-Fos⁺ neurons in these regions in T3Tx-XPro1595 animals (b, c, e, f). We also observed fewer c-Fos⁺, ChAT⁻ interneurons in close proximity to ChAT⁺ (red) SPNs in the T9 IML in T3Tx-XPro1595 animals than T3Tx-saline animals. This indicates that CRD activated fewer sympathetically correlated interneurons near SPNs (g–i). Data are mean ± SEM. *p < 0.05 (Student's t test). ****p < 0.0001 (Student's t test). Scale bar, 50 μm. cc, Central canal.

Inhibiting sTNFα signaling decreases CGRP⁺ afferent sprouting caudal to T3 SCI. a–h, Transverse sections of L6 cord from naive or T3Tx-saline and T3Tx-XPro1595 animals 4 weeks after SCI immunostained for CGRP (n = 4 or 5/group). In naive animals, CGRP⁺ staining was primarily in superficial layers, although some was in the deeper layers of dorsal horn (a, a′). We observed few CGRP⁺ fibers around laminae VII/X (i.e., dorsal gray commissure) (e, e′). There was increased CGRP immunoreactivity in the dorsal horn of T3Tx-saline animals (b, d), particularly in the deeper layers (b′), and around the central canal (f, f′, h). In T3Tx-XPro1595 animals, we observed normal CGRP⁺ fiber density in the dorsal horn (c, c′, d) and laminae VII/X (g, g′, h). i, ELISA analysis of NGF expression in lumbar cord from naive (n = 6) or T3Tx-saline (n = 4) and T3Tx-XPro1595 (n = 5) animals 4 weeks after SCI. T3Tx increased NGF expression, even with XPro1595 treatment. j, k, TrkA protein levels in lumbar cord of T3Tx-saline animals 4 weeks after SCI were lower than in naive animals. Inhibiting sTNFα signaling further diminished TrkA expression. Data are mean ± SEM. *p < 0.05 (one-way ANOVA and post hoc Fisher's LSD test). **p < 0.01 (one-way ANOVA and post hoc Fisher's LSD test). ***p < 0.001 (one-way ANOVA and post hoc Fisher's LSD test). k, The last sample in the blot was an outlier, as determined by the Grubbs' method with α = 0.05, and was excluded from the bar graph in j. Scale bars, 100 μm. cc, Central canal.

sTNFα promotes growth of CGRP⁺ neurites in an NF-κB -dependent manner. Dissociated adult DRGs plated on poly-L-lysine were incubated with just serum-free media or serum-free media containing sTNFα (50 ng/ml), the cell-permeable NF-κB inhibitor SN50 (10 μm), or both sTNFα and SN50 for 48 h. Cultures were stained to visualize CGRP⁺ neurons and their neurites. Compared with the growth seen in the control cultures (a), adding sTNFα to the culture media increases both the total CGRP⁺ growth per neuron (b, e) as well as the length of the longest neurite (f). While the presence of the NF-κB inhibitor SN50 alone did not negatively affect growth compared with control cultures (c, e, f), its presence did nullify the growth-promoting effects of sTNFα (d–f). These data indicate that sTNFα is also capable of directly promoting the growth of CGRP⁺ nociceptive sensory neurons. Data are mean ± SEM. *p < 0.05 (one-way ANOVA and post hoc Fisher's LSD tests). **p < 0.01 (one-way ANOVA and post hoc Fisher's LSD tests). ****p < 0.0001 (one-way ANOVA and post hoc Fisher's LSD tests). n = 30 neurons per condition. Scale bar, 50 μm.