Anat Cell Biol.  2021 Sep;54(3):350-360. 10.5115/acb.20.299.

Ibrutinib reduces neutrophil infiltration, preserves neural tissue and enhances locomotor recovery in mouse contusion model of spinal cord injury

• Torabi S
• Anjamrooz SH
• Zeraatpisheh Z
• Aligholi H
• Azari H

• KMID: 2521048
• DOI: http://doi.org/10.5115/acb.20.299

Abstract

Following acute spinal cord injury (SCI), excessive recruitment of neutrophils can result in inflammation, neural tissue loss and exacerbation of neurological outcomes. Ibrutinib is a bruton’s tyrosine kinase inhibitor in innate immune cells such as the neutrophils that diminishes their activation and influx to the site of injury. The present study evaluated the efficacy of ibrutinib administration in the acute phase of SCI on neural tissue preservation and locomotor recovery. Ibrutinib was delivered intravenously at 3.125 mg/kg either immediately, 12 hours after, or both immediately and 12 hours after SCI induction in adult male C57BL/6 mice. Neutrophil influx into the lesion area was evaluated 24 hours following SCI using light microscopy and immunohistochemistry methods. Animals’ body weight changes were recorded, and their functional motor recovery was assessed based on the Basso mouse scale during 28 days after treatment. Finally, spinal cord lesion volume was estimated by an unbiased stereological method. While animals’ weight in the control group started to increase one week after injury, it stayed unchanged in treatment groups. However, the double injection of ibrutinib led to a significantly lower body weight compared to the control group at 4 weeks post-injury. Mean neutrophil counts per visual field and the lesion volume were significantly decreased in all ibrutinib-treated groups. In addition, ibrutinib significantly improved locomotor functional recovery in all treated groups, especially in immediate and double-injection groups. Neural tissue protection and locomotor functional recovery suggest ibrutinib treatment as a potent immunotherapeutic intervention for traumatic SCI that warrants clinical testing.

Keyword

Spinal cord injuries; Ibrutinib; Recovery of function

Figure

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  Fig. 1 Study design: animals were divided into sham (laminectomy only) and spinal cord injury (SCI) groups. After inducing SCI, animals were treated either with phosphate buffer saline (PBS) (Control) or ibrutinib in PBS; immediately, 12 hours and both immediately and 12 hours after injury (ibrutinib/immediate, ibrutinib/12-hour, ibrutinib/double injection). Neutrophil influx into the lesion area was analyzed 24 hours after injury. Body weight and locomotor function were evaluated every week for 4 weeks. To confirm injury to the spinal cord, immunofluorescent staining against ED1 and glial fibrillary acidic protein were performed at 3 days and 4 weeks after surgery, respectively. Lesion volume was estimated at the end of the study. BMS, Basso mouse scale.

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  Fig. 2 Body weight changes during 4 weeks after SCI. Slight weight loss occurred in all SCI groups one week after injury. While the sham and control groups continued to gain weight starting at one week post-injury, it was plateaued in ibrutinib/immediate and ibrutinib/12-hour groups but significantly decreased in ibrutinib/double injection group at 4 weeks post-SCI. SCI, spinal cord injury. aP<0.05 vs. SCI/vehicle, bP<0.05 vs. Sham.

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  Fig. 3 Neutrophil infiltration analysis 24-hour after SCI: ibrutinib administration significantly decreased neutrophil influx into the injured neural tissue after SCI. Representative images show the infiltrated polymorphonuclear neutrophils at 24 hours post-injury in each group: (A) Sham, (B) Control, (C) Ibrutinib/Immediate, (D) Ibrutinib/12-hour, (E) Ibrutinib/double injection. Neutrophils are visualized as cells with annular and multi-lobulated nuclei (arrows), whereas neuronal cells are distinguished by their single round nuclei (arrowheads). (F) Graph showing mean neutrophil number per field of view. Congo red–Hematoxylin staining. Low to high magnification: 4×, 10×, 20×, 60× objective lens respectively. SCI, spinal cord injury. **P<0.01, aP<0.001 vs. SCI/vehicle, bP<0.0001 sham vs. other groups.

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  Fig. 4 Immunofluorescence based detection of Ly6G positive cells in the lesion site 24 hours after spinal cord injury (SCI): ibrutinib administration significantly decreased Ly6G positive cells in the lesion site 24 hours post-injury. Representative images are provided at the same levels in different groups: (A, B) Sham, (C, D) Control, (E, F) Ibrutinib/Immediate, (G, H) Ibrutinib/12-hour, (I, J) Ibrutinib/double injection, (K) Graph showing mean Ly6G positive cells per field of view. Magnification: upper panel at 4× and lower panel at 100×. Asterisks represent Ly6G positive cells, immunolabeled with red fluorescent Alexa-flour 568 anti-rabbit antibody. ***P<0.001, aP<0.0001 vs. SCI/vehicle, bP<0.0001 sham vs. other groups.

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  Fig. 5 Morphological confirmation of injury in the spinal cord: to confirm injury in the spinal cord, ED1 (CD68) (A–D) and glial fibrillary acidic protein (GFAP) staining (E–H) were performed respectively at 72 hours and 4 weeks after surgery. While ED1 and GFAP are respectively expressed by some resident microglial cells (A, B) and the astrocytes (E, F) in the sham injured cord tissue, their number, and staining intensity increased in the injured cord tissue due to the activation of microglial cells, influx of macrophages (C, D) and reactive astrogliosis (G, H). Magnification: upper panel at 4× and lower panel at 40×. SCI, spinal cord injury.

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  Fig. 6 Hind limb locomotor function after SCI: Ibrutinib administration significantly improved locomotor recovery. The BMS score was estimated pre-operatively, on the first day and every week to assess behavioral recovery following SCI. SCI, spinal cord injury; BMS, Basso mouse scale. **P<0.01, aP<0.05 vs. control.

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  Fig. 7 Total lesion volume after SCI: ibrutinib administration significantly diminished the lesion volume four weeks after SCI. Cavities (arrows) and the surrounding spared tissue were measured in the different groups by Cavalieri᾽s principle. (A) Sham, (B) Control, (C) Ibrutinib/Immediate, (D) Ibrutinib/12-hour, (E) Ibrutinib/double injection. (F) Graph showing total lesion volume in different treatment groups. Cresyl violet staining; magnification: 90×. aP<0.001 vs. control, bP<0.001 vs. other groups.

Reference

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