Influence of intrathecal delivery of bone marrow-derived mesenchymal stem cells on spinal inflammation and pain hypersensitivity in a rat model of peripheral nerve injury

Abstract

Background: Multipotent mesenchymal stem (stromal) cells (MSCs) have been credited with immunomodulative properties, supporting beneficial outcomes when transplanted into a variety of disease models involving inflammation. Potential mechanisms include the secretion of paracrine factors and the establishment of a neurotrophic microenvironment. To test the hypothesis that MSCs release soluble mediators that can attenuate local inflammation, we here analysed the influence of MSCs on the activation of microglia cells, as well as on inflammatory parameters and pain behaviour in a surgical rat model of neuropathic pain.

Methods: We focussed on an experimental model of partial sciatic nerve ligation (PSNL), characterised by a rapid and persistent inflammation in the dorsal lumbar spinal cord where sensory inputs from the sciatic nerve are processed. Via indwelling intrathecal catheters, MSCs were repetitively grafted into the intrathecal lumbar space. Animals were evaluated for mechanical and thermal hypersensitivity over a period of 21 days after PSNL. Afterwards, spinal cords were processed for immunohistochemical analysis of the microglial marker ionized calcium-binding adapter molecule 1 (Iba1) and quantification of inflammatory markers in ipsilateral dorsal horns. We hypothesised that injections on postsurgical days 2 to 4 would interfere with microglial activation, leading to a reduced production of pro-inflammatory cytokines and amelioration of pain behaviour.

Results: PSNL-induced mechanical allodynia or heat hyperalgesia were not influenced by MSC transplantation, and spinal cord inflammatory processes remained largely unaffected. Indeed, the early microglial response to PSNL characterised by increased Iba1 expression in the lumbar dorsal horn was not significantly altered and cytokine levels in the spinal cord at 21 days after surgery were similar to those found in vehicle-injected animals. Grafted MSCs were detected close to the pia mater, but were absent within the spinal cord parenchyma.

Conclusions: We conclude that intrathecal administration is not an appropriate route to deliver cells for treatment of acute spinal cord inflammation as it leads to entrapment of grafted cells within the pia mater. We propose that the early inflammatory response triggered by PSNL in the lumbar spinal cord failed to effectively recruit MSCs or was insufficient to disturb the tissue integrity so as to allow MSCs to penetrate the spinal cord parenchyma.

Figure 1

Experimental design for in vivo experiments. ELISA, enzyme-linked immunosorbent assay; IHC, immunohistochemistry; MSC, mesenchymal stem (stromal) cell; PSNL, partial sciatic nerve ligation.

Figure 2

Modulation of lipopolysaccharide-induced expression of inflammatory cytokines in primary microglial cultures. The relative mRNA expression (normalised to glyceraldehyde 3-phosphate dehydrogenase; GAPDH) of the pro-inflammatory cytokines TNFα (A) and IL-1β (B) was examined in microglial cultures incubated for 48 hours in medium conditioned or not with MSCs and with or without lipopolysaccharide (LPS; 100 ng/ml) during the last 24 hours. Data shown are mean values with SEM from six independent experiments, analysed by two-way analysis of variance followed by Tukey post-hoc test. ^## P < 0.01 and ***/^### P < 0.001. Symbols denote significant differences between LPS-activated and non-activated groups (*) and between control and conditioned medium-treated groups (^#).

Figure 3

Mechanical and thermal hypersensitivity after partial sciatic nerve ligation. Rats were tested for mechanical hypersensitivity (A-C) by using von Frey filaments, and for thermal hypersensitivity (D-E) with the thermal paw stimulator during 20 days after partial sciatic nerve ligation (PSNL) surgery. Paw withdrawal threshold (PWT) of the ipsilateral (A) or contralateral (B) hindpaws after mechanical stimulation are expressed as percentage of the baseline PWT. Paw withdrawal latencies (PWL) of the ipsilateral (D) or contralateral (E) hindpaws after thermal stimulation are expressed as percentage of the baseline PWL. (C,F) Area under the curve (AUC) analysis of the corresponding graphs. Data shown are mean values with SEM. For AUC: *P < 0.05, *** P < 0.001, analysed by two-tailed student’s t-test. Day-by-day differences between groups were calculated by whole sample t-tests with * at least P < 0.05. Each group consisted of 9 to 11 animals.

Figure 4

Effect of intrathecally injected mesenchymal stem cells on mechanical hypersensitivity after partial sciatic nerve ligation. Sham- (A,B) or partial sciatic nerve ligation (PSNL)- (E,F) operated animals either injected with vehicle (“no cells”) or mesenchymal stem cells (MSCs; “cells”) were tested by using von Frey filaments for 20 days after surgery. Paw withdrawal thresholds (PWT) of the ipsilateral (A or E) or contralateral (B or F) hindpaws are expressed as percentage of the baseline PWT. (C,D and G,H) Area under the curve (AUC) analysis of the corresponding graphs, analysed by two-tailed student’s t-test; data shown are mean values with SEM. Day-by-day differences between groups were calculated by whole sample t-tests with * at least P < 0.05. Each group consisted of 9 to 11 animals.

Figure 5

Effect of intrathecally injected mesenchymal stem cells on thermal hypersensitivity after partial sciatic nerve ligation. Sham- (A,B) or partial sciatic nerve ligation (PSNL)- (E,F) operated animals either injected with vehicle (“no cells”) or mesenchymal stem cells (MSCs; “cells”) were tested with the thermal paw stimulator for 20 days after surgery. Paw withdrawal latencies (PWL) of the ipsilateral (A or E) or contralateral (B or F) hindpaws are expressed as percentage of the baseline PWL. (C,D and G,H) Area under the curve (AUC) analysis of the corresponding graphs, analysed by two-tailed student’s t-test; data shown are mean values with SEM. Each group consisted of 9 to 11 animals.

Figure 6

Ionized calcium-binding adapter molecule 1 protein expression 6 days and 21 days after partial sciatic nerve ligation. Immunohistological staining and quantification of the microglial marker ionized calcium-binding adapter molecule 1 (Iba1) at 6 days (A-E) and at 21 days (A’-E’) on the ipsilateral side of the lumbar spinal cord with low and high magnifications. Iba1 staining of sham-operated animals either vehicle-injected (A,A’) or MSC-injected (B,B’) and PSNL animals with vehicle (C,C’) or MSCs (D,D’). Scale bar represents 500 μm. (E,E’) Quantification of the mean grey intensity within the total dorsal horn with SEM, two-way analysis of variance and Tukey post-hoc test for statistical analysis, *P < 0.05 and **P < 0.01. Groups at 6 days after PSNL consisted of 3 to 4 animals and groups at 21 days after PSNL consisted of 5 to 7 animals.

Figure 7

Fate of mesenchymal stem cells after intrathecal injection 6 days and 21 days post-partial sciatic nerve ligation. Immunohistological staining of the lumbar spinal cord (L4/L5) of partial sciatic nerve ligation (PSNL) animals injected with mesenchymal stem cells (MSCs). Bromodeoxyuridine (BrdU)-labelled MSCs appear red and cell nuclei blue. Cells were only validated as BrdU-positive when also showing a blue labelling. (A) Full spinal cord section 6 days after PSNL. Scale bar represents 1000 μm. (B,C) Representative photographs 6 days after PSNL. Scale bar represents 400 μm. (D,E) Representative photographs 21 days after PSNL. Arrows indicate BrdU-positive cells.

Figure 8

Cytokine production in the ipsilateral dorsal horns 21 days post-partial sciatic nerve ligation. Concentrations of (A) IL-1β, (B) IL-6 and (C) IL-10 were measured in supernatants of lysed tissues as derived from the ipsilateral dorsal horns of the L3-L6 spinal cord region. Concentrations of TNFα were below the detection level. Data represent mean with SEM, analysed by two-way analysis of variance followed by Tukey post-hoc test. Each group consisted of 4 to 6 animals. MSC, mesenchymal stem cells; PSNL, partial sciatic nerve ligation.