Transplantation of Neural Precursor Cells Attenuates Chronic Immune Environment in Cervical Spinal Cord Injury

Abstract

Inflammation after traumatic spinal cord injury (SCI) is non-resolving and thus still present in chronic injury stages. It plays a key role in the pathophysiology of SCI and has been associated with further neurodegeneration and development of neuropathic pain. Neural precursor cells (NPCs) have been shown to reduce the acute and sub-acute inflammatory response after SCI. In the present study, we examined effects of NPC transplantation on the immune environment in chronic stages of SCI. SCI was induced in rats by clip-compression of the cervical spinal cord at the level C6-C7. NPCs were transplanted 10 days post-injury. The functional outcome was assessed weekly for 8 weeks using the Basso, Beattie, and Bresnahan scale, the CatWalk system, and the grid walk test. Afterwards, the rats were sacrificed, and spinal cord sections were examined for M1/M2 macrophages, T lymphocytes, astrogliosis, and apoptosis using immunofluorescence staining. Rats treated with NPCs had compared to the control group significantly fewer pro-inflammatory M1 macrophages and reduced immunodensity for inducible nitric oxide synthase (iNOS), their marker enzyme. Anti-inflammatory M2 macrophages were rarely present 8 weeks after the SCI. In this model, the sub-acute transplantation of NPCs did not support survival and proliferation of M2 macrophages. Post-traumatic apoptosis, however, was significantly reduced in the NPC group, which might be explained by the altered microenvironment following NPC transplantation. Corresponding to these findings, reactive astrogliosis was significantly reduced in NPC-transplanted animals. Furthermore, we could observe a trend toward smaller cavity sizes and functional improvement following NPC transplantation. Our data suggest that transplantation of NPCs following SCI might attenuate inflammation even in chronic injury stages. This might prevent further neurodegeneration and could also set a stage for improved neuroregeneration after SCI.

Keywords: apoptosis; chronic inflammation; macrophages; neural precursor cells; neuroregeneration; spinal cord injury; stem cells.

Figure 1

GFP-positive NPCs (green) and Iba1-positive macrophages (red) in the injured spinal cord 8 weeks after traumatic cervical SCI (n = 7). (A) GFP-positive NPCs were mainly distributed in the dorsal white and gray matter (10 × magnification, scale bar = 500 μm). (B) Additionally, GFP-expressing NPCs were often located very close to Iba1-positive macrophages (40 × magnification, scale bar = 15 μm). (C) Surviving NPCs differentiated primarily along the oligodendroglial lineage (GFP/APC), while only a minority of NPCs differentiated into neurons (GFP/NeuN).

Figure 2

Quantification of M1 and M2 activation 8 weeks after SCI (n = 6–7). (A) Pro-inflammatory M1 macrophages were significantly reduced in the NPC group compared to the control group, while anti-inflammatory M2 macrophages were rarely observed in either group. (B–D) Colocalization of Iba1-positive macrophages (red) with the marker enzyme iNOS (green) indicated by the blue arrows confirm the presence of M1 activated macrophages (40 × magnification, scale bar = 40 μm).

Figure 3

(A) Immunodensity of iNOS is in injured control animals (n = 6) maximal at the SCI epicenter with a gradual decline in intensity toward the periphery. (B) Mean iNOS immunodensity was significantly reduced in the NPC group compared to the control group, indicating a reduced pro-inflammatory M1 activation. (C) Immunodensity values for iNOS were especially high in the gray matter with a highly significant group difference in favor of the NPC group.

Figure 4

Quantification of CD-3-positive T lymphocytes 8 weeks after SCI (n = 5). (A) No group differences in perilesional total T cell numbers were observed between NPC and control group. (B–D) Confocal fluorescence microscopy (40 × magnification, scale bar = 15 μm) of the injured spinal cord showing CD-3-positive T lymphocytes (green) co-expressing the nuclear cell marker DAPI (blue).

Figure 5

(A) The quantification of GFAP immunodensity 8 weeks after SCI shows a significant reduction of astrogliosis in the NPC group (n = 5). (B) Confocal fluorescence microscopy (10 × magnification, scale bar = 500 μm) showing GFAP-positive astrocytes (red) with a high density near the cavitation. The outskirts of the astrogliosis, as well as the edge of the cystic cavitation, are shown in yellow.

Figure 6

(A) The intramedullary cavity size, assessed on GFAP-stained cross-sections (see Figure 5B), was reduced in the NPC group compared to control, although this difference did not reach statistical significance (n = 5). (B) The preserved tissue as a percentage of the entire spinal cord volume was higher in the NPC group at all measured time points without reaching statistical significance.

Figure 7

The distribution of caspase-3-positive cells shows an accumulation of apoptotic cells at the epicenter in injured control animals with apoptotic cell numbers steadily decreasing toward the periphery (n = 6). (B) The quantification of caspase-3-positive cell numbers yields a significant reduction in the NPC group compared to the control group (n = 6–7). (C) Apoptotic cell counts were generally higher in the gray matter and were also significantly reduced in the NPC group compared to the control group. (D–F) Caspase-3-positive cells (red) with additional DAPI-expression (blue) were counted as apoptotic cells, as indicated by the yellow arrows (scale bars = 15 μm).

Figure 8

Functional recovery was weekly assessed using the BBB score, the CatWalk XT gait analysis, and the grid walk test (control, n = 9; NPC, n = 13). (A) Although group differences do not reach statistical significance, BBB scores are continuously higher in the NPC group compared to the control group at all observed time points. (B) The density curve, showing the distribution of BBB scores in each group for the first (week 1) and the last (week 8) time point, is clearly clinched toward higher scores in the NPC group. (C) The parameter “Print area” in the CatWalk XT analysis 8 weeks after SCI shows significantly better results (i.e., a larger forelimb area) in the NPC group compared to the control group. (D) The NPC group exhibits higher values in the forelimb parameter “Swing speed” of the CatWalk XT test without reaching statistical significance. (E) NPC-transplanted animals made only marginally fewer step errors in the grid walk test compared to control animals.