Pericytes impair capillary blood flow and motor function after chronic spinal cord injury

Abstract

Blood vessels in the central nervous system (CNS) are controlled by neuronal activity. For example, widespread vessel constriction (vessel tone) is induced by brainstem neurons that release the monoamines serotonin and noradrenaline, and local vessel dilation is induced by glutamatergic neuron activity. Here we examined how vessel tone adapts to the loss of neuron-derived monoamines after spinal cord injury (SCI) in rats. We find that, months after the imposition of SCI, the spinal cord below the site of injury is in a chronic state of hypoxia owing to paradoxical excess activity of monoamine receptors (5-HT₁) on pericytes, despite the absence of monoamines. This monoamine-receptor activity causes pericytes to locally constrict capillaries, which reduces blood flow to ischemic levels. Receptor activation in the absence of monoamines results from the production of trace amines (such as tryptamine) by pericytes that ectopically express the enzyme aromatic L-amino acid decarboxylase (AADC), which synthesizes trace amines directly from dietary amino acids (such as tryptophan). Inhibition of monoamine receptors or of AADC, or even an increase in inhaled oxygen, produces substantial relief from hypoxia and improves motoneuron and locomotor function after SCI.

Figure 1

Trace amines constrict capillaries at pericytes after SCI. (a) Schematic of the spinal cord vasculature. (b) Immunolabeling for pericytes (NG2, p) on a spinal capillary (v) and nearby astrocytes (GFAP, a) caudal to the site of SCI. (c) Schematic of the DIC microscopy set-up used to image capillaries deep below the pial surface of cord after SCI. (d) Top, DIC image of capillary in the spinal cord caudal to a chronic sacral transection, with RBCs (R) and the lumen pseudo-colored red for clarity (arrows point to the endothelium). Bottom, application of tryptophan (30 μM) induced a tonic local vasoconstriction (white arrows; starting 1 min post application) adjacent to a pericyte (p), but not in regions lacking pericytes (black arrows). (e) Same as d, but with AADC inhibitor (NSD1015 [NSD]; 300 μM) applied prior to tryptophan, to prevent tryptamine production. (f) Tryptamine application mimicked tryptophan-induced vasoconstriction (d), adjacent to pericytes (outlined in green). (g) Tryptophan-induced constriction (top, at arrows) reversed by 5-HT_1B receptor antagonist GR127935 (3 μM; bottom). (h) Capillary in normal rat (top), lacking tryptophan-induced constrictions (bottom). (i) Plots of group data for capillary diameters (normalized to pre-drug control) after bath application of amino acids (10, 30, 100 μM tryptophan [trypto10, trypto30, trypto100], 30 μM tyrosine [tyro], 50 μM phenylalanine [phenyl], or 0.1 μM 5-HTP), AADC products (10-100 μM tryptamine [trypta], or 0.3 μM 5-HT) or zolmitriptan (3 and 300 nM: zolm3 and zolm300), with and without inhibition of AADC (NSD1015) or antagonism of the 5-HT_1B and α₂ receptors (with GR127935 and 0.5 μM RX821002, respectively) are shown, and n values are detailed in the Methods. Right plot, arteriole diameter with tryptophan. * P < 0.05: significant change relative to pre-drug control (100%). # P < 0.05 relative change with antagonist or blocker. Box plots and horizontal bar within represent the interquartile range and median, respectively. Error bars extend to the most extreme data point that is within 1.5 times the interquartile range.

Figure 2

AADC, trace amines and 5-HT_1B receptors are co-expressed in pericytes after SCI. (a) Top, immunolabeling with an AADC antibody (black, DAB, upper panel) in a transverse section of a spinal cord caudal to a chronic spinal transection, showing that AADC is widely expressed on capillaries (v), but not arteries (a). Bottom, immunofluorescence for AADC (red) and CD13 (green, pericyte marker) in a lengthwise section of capillary, showing exclusive colocalization of AADC and CD13 in pericytes (p; yellow). (b) Left, DAB immunolabeling of endogenous tryptamine (black) caudal the site of injury, showing dense staining for tryptamine in pericytes (arrows) of capillaries (v), especially in the soma. Inset shows a higher magnification view of a capillary cross-section (scale bar, 10 μm), showing that pericyte (p) cell bodies and processes stain for tryptamine (arrows), but an endothelial cell (e) does not (blue, cresyl-violet stain of the endothelial cell nucleus). Right, immunofluorescent staining for tryptamine (red) and CD13 (green) further showing tryptamine staining in a pericyte. (c) Immunolabeling for the AADC product 5-HT and for the pericyte marker NG2 caudal to a chronic transection injury after pre-treatment with 5-HTP (30 mg/kg, i.p., 25 min prior to fixation). Arrows, staining of 5-HT in NG2-labeled pericytes. (d) This AADC product 5-HT (red) is shown densely accumulated in pericyte cell bodies and processes labelled for AADC (green; at arrows). (e) Schematic of pericyte action on capillaries after SCI, showing diffusion of tryptophan (red) from blood into pericytes, synthesis of tryptamine (yellow) from AADC, and the action of tryptamine on nearby 5HT_1B receptors (blue) to constrict the capillary. (f) Immunolabeling for 5-HT as in panels c and d, but without 5-HTP pre-treatment. (g) Immunolabeling for the 5-HT_1B receptor and NG2 caudal to the site of injury. Arrows show localization of 5-HT_1B receptor on NG2-labelled pericytes, with dense areas of receptor staining on pericyte processes. n = 5 rats tested per condition.

Figure 3

Poor blood flow and hypoxia after chronic SCI. (a) In vivo images of sacral and lumbar spinal cord dorsal vasculature in normal and chronic spinal rats, before and after (20 and 40 s) intracardial injection of methylene blue dye (2% in saline). (b) Perfusion times in sacral (S, caudal to injury) or lumbar (L, rostral) cords of injured and normal uninjured rats, and changes with the 5-HT_1B antagonist GR127935 (GR, 30 μM topically applied to caudal cord) or sodium nitrate (NO donor; see Methods); n = 5 rats per group in box plots. (c) Top, two-photon microscopy image of the in vivo sacral spinal cord vasculature caudal to the site of injury, after FITC-dextran injection (i.v.). The arrow indicates location of a sub-pial spinal capillary imaged. Bottom left, higher magnification and brightened view of the indicated capillary where RBC flow computed. Bottom right, box plots of group capillary RBC flow rate in untreated and NSD1015 (NSD)-treated spinal cords (3 mM topically administered) in chronic spinal rats, n = 5 per group. (d) Schematic of in vivo oxygen measurement (pO₂) in the spinal cord. (e) Low pO₂ caudal to chronic SCI (hypoxia, red), compared to rostral to the SCI or in normal uninjured rats, and changes in pO₂ after dilating vessels with NSD1015 or GR127935 (GR, topical 30 μM), or transient high O₂ breathing (95% O₂, with 5% CO₂ for 1 min). (f) Box plots of pO₂ in uninjured (normal) and injured rats before and after treatments with transient oxygen (95% for 1 min; measured at 10-20 min), GR127935, RX821002 (RX; 5 μM topical), or NSD1015 (NS); n = 5 - 20 per treatment, as detailed in the Methods. Drug effects peaked within minutes and sustained for the duration of the recording 10 - 90 min (peak reported); pO₂ values are means from L4–L6 (rostral to injury or normal lumbar) and S2–S4, Ca1 (caudal to injury or normal sacral) spinal segments. *P < 0.05, significant difference relative to pre-treatment control (f) or normal tissue (b).

Figure 4

Treatments that dilate vessels and improve oxygenation after SCI lead to increased motor activity. (a) Schematic of awake chronic spinal rat in Plexiglas bottle for tail muscle EMG recording and electrical stimulation of the tip of the tail (50×T) to evoke reflexes. (b) Representative muscle activity in a chronic spinal rat, showing baseline EMG activity prior to stimulation (arrow) and long-lasting reflex (LLR) evoked by stimulation, before (top) and after GR127935 treatment (bottom) (intrathecal [i.t.], 30 μl, 10 mM). (c) Box plots of the change in the LLR measured in vitro (rectified-average) induced by blocking the TA-mediated vasoconstriction of capillary flow with either GR127935 (GR, i.p. 8 mg/kg, n = 3 or i.t. 10 mM in 30 μl; n = 2; combined) or RX821002 (RX, i.p. 1 mg/kg, n = 11; or i.t. 3 mM in 30 μl, n = 5), and compared to the change in LLR studied in the isolated in vitro spinal cord, with application of GR127935 (3 μM, n = 18), RX821002 (1 μM, n = 42) and NSD1015 (NS, 300 μM, n = 9) (see Supplementary-Fig-15). LLR normalized to LLR prior to drugs, 100%. (d) Representative EMG trace in a chronic spinal rat before and 15 min after transient breathing of 95% O₂ (1 min, with 5% CO₂). Smoothed rectified rhythmic activity is indicated by the blue line. (e) Time course of mean LLR before and after transiently increased O₂ breathing in vivo (1 min), compared to LLR response to increased O₂ in the isolated in vitro spinal cord (pO₂ increased in nACSF). (f) Box plots of LLR in injured rats in vivo, either prior to treatment (Pre), during treatment with O₂ or CO₂, or 10–20 min post-treatment (post). Treatments were transient hyperoxia (O₂, 95% for 1 min, n = 8) or hypercapnia (CO₂, 10% in air for 30 sec, n = 10) breathing. Also shown are data for in vitro isolated spinal cords during or after treatment with O₂ (n = 6). All conditions normalized as in c. * P < 0.05 significant difference relative to pre-drug condition.

Figure 5

Thoracic contusion or staggered hemisection injury induces chronic hypoxia that impairs locomotion. (a) Schematic of thoracic staggered-hemisection SCI, which transects all descending axons from the brain, including those containing monoaminergic neurons. (b) Video image sequence of a rat walking one month after receiving a staggered hemisection injury. Impaired hindlimb function while walking is evident from poor weight support (quantified as torso height above ground), leg extensor spasms (quantified as spasm time relative to step-cycle duration), slow steps (number of hindlimb plantar steps per step-cycle of front leg) and poor foot placement (caudal to behind hip). Hip (iliac crest), knee and ankle joints are shown with dots and lines. Arrow shows foot movement. (c) Effects of intrathecal application of GR127935 (10 mM in 30 μl) on locomotion, with locomotion phases annotated above. (d) Plots of mean locomotor parameters for each rat (circles) with staggered hemisection injury (3 - 5 weeks post injury), including body height, extensor spasm time, number of successful hindlimb steps and foot placement error, before and after application of GR127935 or transient breathing of 95% O₂ (90 s; with 5% CO₂; measured 10–15 min post treatment). n = 7 rats per treatment group. Error bars: s.e.m, bars: group means (e) Plots of mean locomotor parameters for each rats with contusion injury, in a similar format as d. Effects 1 and 24 h after treatment with NSD1015 (100 mM in 30 μl i.t.; NSD) were also measured. n = 9 rats for GR127935 and oxygen, and n = 6 for NSD. (f-g) Plots of pO₂ in uninjured (normal) rats (lumbar cord) and in SCI rats (caudal [lumbar] and rostral [thoracic] to the site of injury), before and 10-15 min after treatments with transient oxygen (95%, 90 s), GR127935 (GR, topically applied 30 μM), or RX821002 (RX, topical 5 μM). *P < 0.05: difference relative to normal cord or change with treatment.