Potential neuroprotective effect of Anakinra in spinal cord injury in an in vivo experimental animal model

Abstract

Objective: To evaluate the therapeutic effects of inhibiting interleukin-1 beta (IL-1 beta) in vivo using Anakinra in an experimental model of spinal cord injury (SCI).

Methods: All experimental procedures were performed in the animal laboratory of Ankara Education and Research Hospital, Ankara, Turkey between August 2012 and May 2014. The SCI was induced by applying vascular clips to the dura via a 4-level T5-T8 laminectomy. Fifty-four rats were randomized into the following groups: controls (n = 18), SCI + saline (n = 18), and SCI + Anakinra (n = 18). Spinal cord samples were obtained from animals in both SCI groups at one, 6, and 24 hours after surgery (n = 6 for each time point). Spinal cord tissue and serum were extracted, and the levels of IL-1 beta, malondialdehyde, glutathione peroxidase, superoxide dismutase, and catalase were analyzed. Furthermore, histopathological evaluation of the tissues was performed.

Results: The SCI in rats caused severe injury characterized by edema, neutrophil infiltration, and cytokine production followed by recruitment of other inflammatory cells, lipid peroxidation, and increased oxidative stress. After SCI, tissue and serum IL-1 beta levels were significantly increased, but were significantly decreased by Anakinra administration. Following trauma, glutathione peroxidase, superoxide dismutase, and catalase levels were decreased; however, Anakinra increased the activity of these antioxidant enzymes. Malondialdehyde levels were increased after trauma, but were unaffected by Anakinra. Histopathological analysis showed that Anakinra effectively protected the spinal cord tissue from injury.

Conclusion: Treatment with Anakinra reduces inflammation and other tissue injury events associated with SCI.

Figure 1

Tissue biochemical alterations in the 3 groups. Values are expressed as mean ± SD or median (interquartile range), where applicable. Times indicate the timepoint after injury in hours. CAT - catalase, GPx - glutathione peroxidase, MDA - malondialdehyde, SOD - superoxide dismutase

Figure 2

Serum biochemical alterations in the 3 groups. Values are expressed as mean ± SD or median (interquartile range), where applicable. Times indicate the timepoint after injury in hours. CAT - catalase, GPx - glutathione peroxidase, MDA - malondialdehyde, SOD - superoxide dismutase

Figure 3

Photomicrograph of the control group. The parenchymal features of the white (WM) and surrounding gray matter, neuronal morphology, and vascular structures appeared normal in the control group (Hematoxylin & Eosin staining ×4). PH - posterior horn, CC - central canal, AH - anterior horn

Figure 4

Photomicrographs of the trauma group at all time points. A) There was mild to moderate edema, and prominent disturbances were seen between boundaries of white (WM) and gray matter (GM), central canal (CC), and hemorrhagic areas in GM (arrows) (Hematoxylin & Eosin [H&E] staining, ×4). Inlet showing higher magnification of hemorrhagic areas (arrows) (H&E ×40) B) Hemorrhagic areas in GM (arrows), cavitation (double arrows), and dilatation and edema in WM fiber bundles (stars) (H&E ×10). C) Serious neuronal loss and hemorrhagic areas (H&E ×10). Upper left inlet showing hemorrhage (E) and spongiosis (stars) in GM (H&E ×40). Lower right inlet showing edema (stars), axonal degeneration and dilatation of WM fiber bundles (arrows) (H&E ×40).

Figure 5

Photomicrographs of the Anakinra-treated group one hour after trauma showing A) regions and structure of spinal cord gray matter (GM), white matter (WM), central canal (CC), multipolar neurons (MNs) (Hematoxylin & Eosin [H&E] staining ×4). B) Edema, axonal dilatation and degeneration in WM (stars), and hemorrhagic areas in GM (arrows), (H&E ×10). C-D) Swollen astrocytic processes forming clear areas around degenerated neurons (arrowheads), axonal dilatation and degeneration (stars), and central cavitation around CC (double arrows)(H&E ×10). bv - blood vessel, P - pia mater

Figure 6

Photomicrographs of the Anakinra-treated group at 6 hours after trauma showing less edema and fewer degenerated neurons showing: A) central cavitation (arrowhead, Hematoxylin & Eosin staining [H & E] ×10) B) Eosinophilic neuron (thick arrow, H & E ×40). P - pia mater, GM - gray matter, WM - white matter

Figure 7

Photomicrograph of the Anakinra-treated group at 24 hours after trauma showing less edema and neurons (stars) with intensely stained basophilic cytoplasm in gray matter (GM), (Hematoxylin & Eosin [H&E] staining ×4). Inlet showing axons with myelin sheath (arrow), degenerated axons and dilated fibers (stars) (H&E ×40).