Transplant restoration of spinal cord inhibitory controls ameliorates neuropathic itch

Abstract

The transmission of pruritoceptive (itch) messages involves specific neural circuits within the spinal cord that are distinct from those that transmit pain messages. These itch-specific circuits are tonically regulated by inhibitory interneurons in the dorsal horn. Consistent with these findings, it has previously been reported that loss of GABAergic interneurons in mice harboring a deletion of the transcription factor Bhlhb5 generates a severe, nonremitting condition of chronic itch. Here, we tested the hypothesis that the neuropathic itch in BHLHB5-deficient animals can be treated by restoring inhibitory controls through spinal cord transplantation and integration of precursors of cortical inhibitory interneurons derived from the embryonic medial ganglionic eminence. We specifically targeted the transplants to segments of the spinal cord innervated by areas of the body that were most severely affected. BHLHB5-deficient mice that received transplants demonstrated a substantial reduction of excessive scratching and dramatic resolution of skin lesions. In contrast, the scratching persisted and skin lesions worsened over time in sham-treated mice. Together, these results indicate that cell-mediated restoration of inhibitory controls has potential as a powerful, cell-based therapy for neuropathic itch that not only ameliorates symptoms of chronic itch, but also may modify disease.

Figure 3. Transplanted MGE progenitor cells differentiate into GABAergic interneurons and integrate into host circuitry of the Bhlhb5 mutant mice.

(A–C) GFP-expressing (green) MGE cells in the spinal cord dorsal horn of Bhlhb5 mutant mice. Cells dispersed throughout the dorsal horn (A), extended long processes (B), and the great majority expressed the neuronal marker NeuN (C). DC, dorsal column. (D–F) Most of the GFP⁺ cells also express GABA. (G–I) Some GFP⁺ cells coexpress SST, indicating that the MGE transplants retain characteristics of cortical GABAergic interneurons. (J–L) A peripheral noxious stimulus (hind paw injection of dilute formalin) induces expression of Fos, a marker of neuronal activity, in GFP+ (i.e., transplanted) cells. Arrows point to double-labeled cells. Inset in L illustrates 2 GFP⁺Fos⁺ double-labeled cells. Scale bar: 150 μm (A); 50 μm (B–L); 25 μm (inset).

Figure 2. Intraspinal transplantation of GABAergic precursor (MGE) cells reduces excessive scratching, biting, and licking in Bhlhb5 mutant mice.

Bhlhb5 mutant mice exhibit excessive licking, biting, and scratching (baseline). This spontaneous behavior usually developed around 4 weeks of age. Two weeks after transplantation, these behaviors were significantly reduced in mice that received MGE cells (red, n = 6; –70%, *P < 0.05). In contrast, these behaviors persisted in control animals (black, n = 7; –12% NS). Scratching behavior was measured in minutes over a 30-minute period of observation. The illustration to the right displays the individual data points.

Figure 1. Intraspinal transplantation of GABAergic precursor (MGE) cells resolves skin lesions in Bhlhb5 mutant mice.

(A–F) Two representative examples of Bhlhb5 mutant mice before (A and D), 2 weeks after (B and E), and 5 weeks after (C and F) MGE transplantation into the lumbar (A–C) and cervical spinal cord (D–F). There is remarkable, progressive improvement of the affected region ipsilateral to the transplant. In some cases, we observed new hair growth in previously denuded regions (arrow in C). In contrast, skin lesions in animals that received only medium (G–I) progressively worsened. All mice were 5 to 6 weeks of age at the time of transplant. Because of the deterioration of control animals, euthanization was generally required by 7 to 8 weeks of age.