Synaptic plasticity and sensory-motor improvement following fibrin sealant dorsal root reimplantation and mononuclear cell therapy

Abstract

Root lesions may affect both dorsal and ventral roots. However, due to the possibility of generating further inflammation and neuropathic pain, surgical procedures do not prioritize the repair of the afferent component. The loss of such sensorial input directly disturbs the spinal circuits thus affecting the functionality of the injuried limb. The present study evaluated the motor and sensory improvement following dorsal root reimplantation with fibrin sealant (FS) plus bone marrow mononuclear cells (MC) after dorsal rhizotomy. MC were used to enhance the repair process. We also analyzed changes in the glial response and synaptic circuits within the spinal cord. Female Lewis rats (6-8 weeks old) were divided in three groups: rhizotomy (RZ group), rhizotomy repaired with FS (RZ+FS group) and rhizotomy repaired with FS and MC (RZ+FS+MC group). The behavioral tests electronic von-Frey and Walking track test were carried out. For immunohistochemistry we used markers to detect different synapse profiles as well as glial reaction. The behavioral results showed a significant decrease in sensory and motor function after lesion. The reimplantation decreased glial reaction and improved synaptic plasticity of afferent inputs. Cell therapy further enhanced the rewiring process. In addition, both reimplanted groups presented twice as much motor control compared to the non-treated group. In conclusion, the reimplantation with FS and MC is efficient and may be considered an approach to improve sensory-motor recovery following dorsal rhizotomy.

Keywords: dorsal root rhizotomy; fibrin sealant; mononuclear cells; motor control; sensory recovery.

Figure 1

Dorsal view of the lumbar spinal cord at L4-L6 level, following hemi-laminectomy. (A) Lumbar intumescence showing intact L4–L6 dorsal roots. (B) Animal without reimplantation. The arrow shows one root disconnected from the spinal cord. (C) Animal subjected to reimplantation with fibrin sealant. The arrows (L6, L5, and L4, respectively) show the exact point of rhizotomy. Scale bar = 1 mm.

Figure 2

Walking track test carried out using the Catwalk system (Noldus Inc., Holland). (A) Peroneal functional index. (B) Max contact max intensity. (C) Print area. (D) Regularity index. (E) Stand (time in seconds that the animal stood over the paw ipsilateral to the lesion/repaired side). (F) Max contact area. The vertical dotted lines represent the moment of lesion/repair. CL, contralateral; FS, fibrin sealant; IL, ispsilateral; PT, pretest; MC, mononuclear cells; RZ, rhizotomy. ^***p < 0.001.

Figure 3

Electronic von-Frey measurements (average values) obtained from the right hind paw (lesioned). The values are shown in grams (g) applied to trigger the “flinch” response. Statistical differences among groups are indicated with brackets. Asterisks above each dot represent the comparison between the time point and the respective pre-test. FS, fibrin sealant; PT, pretest; MC, mononuclear cells; RZ, rhizotomy.

Figure 4

Immunohistochemistry for glial fibrillary acidic protein (GFAP). (A–L) Representative images of RZ, RZ+FS and RZ+FS+MC, 1, 4, and 8 weeks post lesion. (M) Quantification (ratio IL/CL) of the integrated density of pixels, obtained in the region delimited by dashed lines. (N) Representation of laminae I and II (total quantification area: 3.9 × 10⁵ μm²). CL, contralateral; FS, fibrin sealant; IL, ispsilateral; MC, mononuclear cells; RZ, rhizotomy. Scale bar = 50 μm. ^*p < 0.05; ^**p < 0.01.

Figure 5

Immunohistochemistry for glial fibrillary acidic protein (GFAP). (A–L) Representative images of RZ, RZ+FS and RZ+FS+MC, 1, 4, and 8 weeks post lesion. (M) Quantification (ratio IL/CL) of the integrated density of pixels, obtained in the region delimited by dashed circles. (N) Representation of lamina IX (total quantification area: 8.5 × 10⁵ μm²). CL, contralateral; FS, fibrin sealant; IL, ispsilateral; MC, mononuclear cells; RZ, rhizotomy. Scale bar = 50 μm. ^**p < 0.01; ^***p < 0.001.

Figure 6

Immunohistochemistry for ionized calcium binding adaptor protein (Iba1). (A–L) Representative images of RZ, RZ+FS and RZ+FS+MC, 1, 4, and 8 weeks post lesion. (M) Quantification (ratio IL/CL) of the integrated density of pixels, obtained in the region delimited by dashed lines. (N) Representation of laminae I and II (total quantification area: 3.9 × 10⁵ μm²). CL, contralateral; FS, fibrin sealant; IL, ispsilateral; MC, mononuclear cells; RZ, rhizotomy. Scale bar = 50 μm. ^*p < 0.05; ^**p < 0.01; ^***p < 0.001.

Figure 7

Immunohistochemistry for ionized calcium binding adaptor protein (Iba1). (A–L) Representative images of RZ, RZ+FS and RZ+FS+MC, 1, 4, and 8 weeks post lesion. (M) Quantification (ratio IL/CL) of the integrated density of pixels, obtained in the region delimited by dashed circles. (N) Representation of lamina IX (total quantification area: 8.5 × 10⁵ μm²). CL, contralateral; FS, fibrin sealant; IL, ispsilateral; MC, mononuclear cells; RZ, rhizotomy. Scale bar = 50 μm. ^*p < 0.05.

Figure 8

Immunohistochemistry for vesicular glutamate transporter 1 (VGLUT1). (A–L) Representative images of RZ, RZ+FS and RZ+FS+MC, 1, 4, and 8 weeks post lesion. (M) Quantification (ratio IL/CL) of the integrated density of pixels, obtained in the region delimited by dashed lines. (N) Representation of lamina III (total quantification area 3.9 × 10⁵ μm²). CL, contralateral; FS, fibrin sealant; IL, ispsilateral; RZ, rhizotomy; MC, mononuclear cells. Scale bar = 50 μm. ^*p < 0.05; ^**p < 0.01; ^***p < 0.001.

Figure 9

Immunohistochemistry for vesicular glutamate transporter 1 (VGLUT1). (A–L) Representative images of RZ, RZ+FS and RZ+FS+MC, 1, 4, and 8 weeks post lesion. (M) Quantification (ratio IL/CL) of the integrated density of pixels, obtained in the region delimited by dashed circles. (N) Representation of lamina IX (total quantification area 8.5 × 10⁵ μm²). CL, contralateral; FS, fibrin sealant; IL, ispsilateral; MC, mononuclear cells; RZ, rhizotomy. Scale bar = 50 μm. ^**p < 0.01.

Figure 10

(A–D) Immunohistochemistry for VGLUT1. (A) Contralateral side. (B) IL side of RZ alone group. (C) IL side of RZ+FS group. (D) IL side of RZ+FS+MC group. White dotted ellipse represents the injury/reimplantation site. Orange dotted circle represents lamina IX. (E–G) Schematic representation of the proposed regeneration process following dorsal root reimplantation with fibrin sealant associated with mononuclear MC therapy. (E) RZ group. (F) RZ+FS group. (G) RZ+FS+SC group. FS, fibrin sealant; IL, ispsilateral; MC, mononuclear cells; RZ, rhizotomy. Scale bar = 500 μm.

Figure 11

Immunohistochemistry for GAP-43. (A–L) Representative images of RZ, RZ+FS and RZ+FS+MC, 4 weeks post lesion. The location at the dorsal root area (dashed circles) and within the spinal cord (dashed lines) is indicated by the schematic drawings. Observe the increased number of positive axons toward deeper laminae following reimplantation of dorsal roots associated with cell therapy. FS, fibrin sealant; IL, ispsilateral; MC, mononuclear cells; RZ, rhizotomy. Scale bar = 50 μm.

Figure 12

Immunohistochemistry for GAD65. (A–L) Representative images of RZ, RZ+FS and RZ+FS+MC, 1, 4, and 8 weeks post lesion. (M) Quantification (ratio IL/CL) of the integrated density of pixels, obtained in the region delimited by dashed lines. (N) Representation of lamina III (total quantification area 3.9 × 10⁵ μm²). CL, contralateral, FS, fibrin sealant; IL, ispsilateral; MC, mononuclear cells; RZ, rhizotomy. Scale bar = 50 μm. ^*p < 0.05; ^**p < 0.01.