Improvement of Contused Spinal Cord in Rats by Cholinergic-like Neuron Therapy

Abstract

Background: Disability in spinal cord injury is an important medical problem, and cell transplantation is considered as an option for the treatment.

Objectives: The purpose of this study is to use bone marrow stromal cells (BMSCs) derived cholinergic neuron-like cells (CNL) in order to ameliorate the contusion model of spinal cord injury in rats.

Materials and methods: The CNLs were produced by pre inducing BMSCs with β-mercaptoethanol (BME) followed by inducing with nerve growth factor (NGF). The cells were immunoreactive to neurofilament 200, NeuN, synaptophysin, synapsin, microtubule associated protein-2 and choline acetyl transferase (ChAT). The CNL were transplanted in contused rats (CR), which were sacrificed after 12 weeks.

Results: The results showed that BBB test showed an improvement in the CR, while the quantitative analysis showed that the improvement rate was higher in the rats treated with CNL than those treated with BMSCs only or the untreated animals, similar results were noticed in the improvement index. Immunohistochemical analysis of the tissue section prepared from the CR showed that the transplanted cells were engrafted and integrated in the traumatized spinal cord. The morphometric analysis showed that the volume density of the cavity in the CNL treated rats was significantly lower than that of the untreated ones, while the spinal tissue regeneration index was significantly higher.

Conclusions: The conclusion of the study is that CNL can improve the injured spinal cord.

Keywords: Cholinergic Neurons; Contusions; Spinal Cord Injuries; Tissue Therapy.

Figure 1. Phase Contrast Photomicrographs from Bone Marrow Stromal Cells( BMSCs) with Different Preparations (A-D) and Immunostaining of BMSCs with Different Markers in order to Characterize These Cells (E and F)

Figure 2. Electrophogram of Oct-4 in the Adult Spinal Cord (a differentiated tissue) Used as Negative Control

Figure 3. Immunostaining of Bone Marrow Stromal Cells ( BMSCs) With Anti-Fibronectin Antibody A), and Pre induced and Induced BMSCs with Anti-neurofilament (NF) Antibodies (NF-200: C and E; and NF-68: B and D) and Induced BMSCs Labeled with BrdU (F)

Figure 4. Immunostaining of Bone Marrow Stromal Cells (BMSCs) with Anti-Synaptophysin (A and C), Anti-MAP2 (B and E), and Anti-choline Acetyl Transferase (ChAT: D and F)

Figure 5. A represents a histogram of BBB test for the sham-operated (solid white), the negative control group injected without treatment (brick pattern), the negative control group injected with normal saline (dotted pattern), the animal group treated with bone marrow stromal cells (vertical lines pattern) and the animal group treated with cholinergic neuron-like cells (horizontal lines pattern)

Figure 6. Shows the histograms of the percentages of immunoreactive cells (PIC) to nestin, synaptophysin, NeuN and ChAT (A), and the numerical density per area of the transplanted trans differentiated cholinergic neuron-like cells (B)

Figure 7. Immunostaining of the induced BMSCs into cholinergic neuron phenotypes labeled with bromodeoxyuridine (BrdU) and transplanted in the rats' contused spinal cord, the animals are sacrificed after 3 months

Figure 8. A) Cholinergic neuron phenotypes and endogenousChAT positive cells simultaneously labeled with mouse anti-ChAT monoclonal antibody (primary antibody) and incubated with (secondary antibody) rabbit anti-mouse antibody conjugated with FITC. B) Cholinergic neuron phenotypes labeled with mouse anti-BrdU monoclonal antibody (primary antibody) and incubated with rabbit anti-mouse antibody conjugated with Rhoda mine. C) The images A and B merged showing a double labeled transplanted cell (arrow), empty arrowhead represents a cell immunostained with anti-ChAT antibody which is the host neuron immunoreactive to anti-ChAT antibody, and arrowhead presents a non-cholinergic neuronal transplanted cell. D) A phase contrast image of A, B and C (scale bar = 50 μm)