Predifferentiated GABAergic neural precursor transplants for alleviation of dysesthetic central pain following excitotoxic spinal cord injury

Abstract

Intraspinal quisqualic acid (QUIS) injury induce (i) mechanical and thermal hyperalgesia, (ii) progressive self-injurious overgrooming of the affected dermatome. The latter is thought to resemble painful dysesthesia observed in spinal cord injury (SCI) patients. We have reported previously loss of endogenous GABA immunoreactive (IR) cells in the superficial dorsal horn of QUIS rats 2 weeks post injury. Further histological evaluation showed that GABA-, glycine-, and synaptic vesicular transporter VIAAT-IR persisted but were substantially decreased in the injured spinal cord. In this study, partially differentiated GABA-IR embryonic neural precursor cells (NPCs) were transplanted into the spinal cord of QUIS rats to reverse overgrooming by replenishing lost inhibitory circuitry. Rat E14 NPCs were predifferentiated in 0.1 ng/ml FGF-2 for 4 h prior to transplantation. In vitro immunocytochemistry of transplant cohort showed large population of GABA-IR NPCs that double labeled with nestin but few colocalized with NeuN, indicating partial maturation. Two weeks following QUIS lesion at T12-L1, and following the onset of overgrooming, NPCs were transplanted into the QUIS lesion sites; bovine adrenal fibroblast cells were used as control. Overgrooming was reduced in >55.5% of NPC grafted animals, with inverse relationship between the number of surviving GABA-IR cells and the size of overgrooming. Fibroblast-control animals showed a progressive worsening of overgrooming. At 3 weeks post-transplantation, numerous GABA-, nestin-, and GFAP-IR cells were present in the lesion site. Surviving grafted GABA-IR NPCs were NeuN(+) and GFAP(-). These results indicate that partially differentiated NPCs survive and differentiate in vivo into neuronal cells following transplantation into an injured spinal cord. GABA-IR NPC transplants can restore lost dorsal horn inhibitory signaling and are useful in alleviating central pain following SCI.

Keywords: GABA; VIAAT; cortical progenitor cell; neuropathic pain; overgrooming; quisqualic acid; spinal cord injury; transplantation.

Figure 1

Overgrooming lesions were primarily observed on at-level ipsilateral dermatomes two weeks after lesion [(A): black arrow]. (B) A 3D model of spinal cord showing three injections of approximate 1 mm in depth from the dorsal surface needed to induce overgrooming in QUIS animals. (C) Characteristic loss of spinal neurons (Neu-N immunoreactivity) in the neck of dorsal horn 2 weeks after QUIS injection (white arrow). Neurons from the superficial lamina I and II are mostly spared from the excitotoxic cell death.

Figure 2

Decreased immunoreactivity for inhibitory signaling components in injured spinal cord gray matter. Spinal cord sections were stained for GABA (A,B) and glycine (C,D). Uninjured dorsal horns showed robust staining for both neurotransmitters (A,C), but staining was decreased in injured dorsal horns (B,D). Spinal cord sections were also triple-stained for GABA, VIAAT, and synaptobrevin. A region within the uninjured dorsal horn was enlarged (E), showing extensive VIAAT labeling (arrowheads), few lone GABA puncta (arrows), and numerous triple-labeled white puncta (block arrows). Enlargement of injured dorsal horn (F) revealed substantially decreased VIAAT, abundant free GABA, and few triple-labeled puncta. Scale bars = 200 μm (D); 50 μm (F).

Figure 3

Brief exposure to varying concentrations of FGF-2 induces differentiation of embryonic cortical progenitor cells into GABAergic phenotype in cultured neurospheres. Progenitor cells exposed to low concentration of FGF-2 expressed the largest number of GABA-IR cell (A,B). Majority of these cells were found in or adjacent to the neurospheres and did not show migratory pattern. Progenitor cells exposed to 1 or 10 ng/ml FGF-2 did not display as many GABA-IR cells as those exposed to 0.1 ng/ml. These GABA-IR cells were found predominantly outside the neurospheres radiating outwardly in a migratory pattern (C,D). Large proportion of NPCs can be labeled with BrdU, however, the GABA-IR cells did not co-label with BrdU (E). A high magnification light microscopy image of GABA-IR cells inside a neurosphere labeled with DAB peroxidase reaction (F). Scale bars = 20μm (A); 50μm (B–E).

Figure 4

Predifferentiated embryonic precursor neurospheres expressed robust immunoreactivity for GABA and other marker for cell differentiation 5–7 days after harvest. (A) Many of GABA-IR NPCs colocalized with GAD65/67-IR cells (arrows). (B) Majority of predifferentiated GAD65/67-IR NPCs also expressed NeuN-IR. (C) Majority of predifferentiated GABA-IR NPCs also expressed nestin-IR; Not every nestin-IR cells expressed GABA-IR. Many GABA-IR cells colocalized and expressed NeuN-IR (D), β-III-tubulin-IR (E), and MAP-2-IR (F).

Figure 5

Photomicrographs showing changes in size of overgrooming in QUIS animals transplanted with control bovine fibroblast cells [pretransplantation: (A) 3 weeks post: (C)] and predifferentiated GABA-IR NPCs [pretransplantation: (B), 3 weeks post: (D)].

Figure 6

Percent change of the overgrooming area from pretransplantation for GABA and fibroblast-transplanted rats. Control animals displayed significant increases in total overgrooming area (A) and percent change of overgrooming area (B) post-transplantation vs. pretransplantation and also compared to GABA-transplanted animals. GABA-transplanted rats did not display statistically significant changes in either total or percent change of overgrooming compared to pretransplantation. *p < 0.05 vs. pretransplantation, ^#p < 0.05 vs. GABA group.

Figure 7

Transplanted predifferentiated GABA-IR NPCs were visible in the spinal cord of QUIS animals three weeks after transplantation, and showed varying densities in the host spinal cord. Animals that stopped overgrooming [healed; (A,B)] or displayed reduction in overgrooming (C,D) exhibited large number of transplanted GABA-IR NPCs in the spinal gray matter. Animals that continued to groom contained very few number of surviving GABA-IR cells in the spinal cord (E,F). Scale bars = 500 μm.

Figure 8

Photomicrographs of QUIS spinal cord transplanted with GABA-IR NPCs. The NPCs were double labeled with NeuN (A–D), nestin (E–H), GFAP (I–L), and β-III-tubulin (M–P). Medium (A,E,I,M) and high magnification (B,F,J,N) of QUIS spinal cord stained for GABA-IR NPCs. The dotted lines represent the spinal gray matter, and white boxes represent the ROI in panels (B,F,J,N). Majority of GABA-IR NPCs double labeled with NeuN-IR (B–D). However, the GABA-IR NPCs did not colocalize with nestin (F–H), GFAP (J–L), or with β-III-tubulin (N–P).