Guanosine reduces apoptosis and inflammation associated with restoration of function in rats with acute spinal cord injury

Abstract

Spinal cord injury results in progressive waves of secondary injuries, cascades of noxious pathological mechanisms that substantially exacerbate the primary injury and the resultant permanent functional deficits. Secondary injuries are associated with inflammation, excessive cytokine release, and cell apoptosis. The purine nucleoside guanosine has significant trophic effects and is neuroprotective, antiapoptotic in vitro, and stimulates nerve regeneration. Therefore, we determined whether systemic administration of guanosine could protect rats from some of the secondary effects of spinal cord injury, thereby reducing neurological deficits. Systemic administration of guanosine (8 mg/kg per day, i.p.) for 14 consecutive days, starting 4 h after moderate spinal cord injury in rats, significantly improved not only motor and sensory functions, but also recovery of bladder function. These improvements were associated with reduction in the inflammatory response to injury, reduction of apoptotic cell death, increased sparing of axons, and preservation of myelin. Our data indicate that the therapeutic action of guanosine probably results from reducing inflammation resulting in the protection of axons, oligodendrocytes, and neurons and from inhibiting apoptotic cell death. These data raise the intriguing possibility that guanosine may also be able to reduce secondary pathological events and thus improve functional outcome after traumatic spinal cord injury in humans.

Fig. 1

Open field walking test (OFWT) scores from day 0 (the same day as surgery, prior to treatment) to 28 days after spinal cord injury for groups of saline- and guanosine-treated animals (means ± SEM). Animals with normal spinal cord function score 21, whereas a score of 0 represents total paralysis. Hind limb locomotor function in guanosine-treated animals (n = 11) was significantly better than in rats that received saline treatment (n = 10; P < 0.01) from day 1 to 28 after injury

Fig. 2

Hind limb placing response (HLPR) scores from day 0 (the same day as surgery, prior to treatment) to 28 days after spinal cord injury for groups of saline- and guanosine-treated animals (means ± SEM). Animals with normal spinal cord function score 2, whereas a score of 0 represents total paralysis. Compared with saline-treated control animals (n = 10), guanosine-treated animals (n = 11) had a significantly better improvement of their hind limb placing responses (P < 0.01) from day 1 to 28 after injury

Fig. 3

Foot orienting response (FOR) scores from day 0 (the same day as surgery, prior to treatment) to 28 days after spinal cord injury for groups of saline- and guanosine-treated animals (means ± SEM). Animals with normal spinal cord function score 2, whereas a score of 0 represents total paralysis. Compared with saline-treated control animals (n = 10), guanosine-treated animals (n = 11) had a significantly better improvement of their foot orienting responses (P < 0.01) from day 1 to 28 after injury

Fig. 4

Inclined plane test (IPT) scores from day 0 (the same day as surgery, prior to treatment) to 28 days after spinal cord injury for groups of saline- and guanosine-treated animals (means ± SEM). Compared with saline-treated control animals (n = 10), guanosine-treated animals (n = 11) had a significantly better score in their inclined plane test (P < 0.05) from day 1 to 28 after injury

Fig. 5

Post-lesional sensitivity of hind limbs touched by a hot plate from day 0 (the same day as surgery, prior to treatment) to 28 days after spinal cord injury for groups of saline- and guanosine-treated animals. Values are the means ± SEM of the average time of withdrawal of left and right hind paws during contact with a hot plate. Compared with saline-treated control animals (n = 10), guanosine-treated animals (n = 11) had a significantly better improvement of their sensory function (*P < 0.05) from day 2 to 14 after injury

Fig. 6

Time course of recovery of lower urinary tract function (spontaneous voiding). Urinary bladders were expressed every 12 h, and the collected urine volume was measured. Means ± SEM of the volume for each group. Compared with saline-treated control animals (n = 10), guanosine-treated animals (n = 11) had significantly less urine collected with time after SCI (*P < 0.05; **P < 0.001). The rats that received guanosine had empty bladders by day 7 after injury

Fig. 7

Fluorescent immunostaining using antibodies against a marker (ED-1) for macrophages and activated microglia in cross sections of cords from saline-treated (a) and guanosine-treated (b) animals at the lesion site 4 weeks after injury. There were fewer ED-1-immunolabeled cells in cords of guanosine-treated rats (b, e) compared to the cords of vehicle-treated rats (a, e; P < 0.01). GFAP-immunofluorescent staining of cross sections of spinal cords from vehicle-treated (c) and guanosine-treated (d) animals at the lesion site 4 weeks after injury showed no difference in immunostaining of GFAP between the cords of guanosine-treated rats (d) and vehicle-treated rats (c). Scale bar = 50 μm for all

Fig. 8

TUNEL-positive apoptotic cells in spinal cord lesions were quantified by counting the total number of TUNEL-positive nuclei through entire cross sections. Compared with the cords from saline-treated animals (a, c), guanosine-treated cords had significantly fewer TUNEL-positive cells (b, c; P < 0.001). Scale bar = 50 μm for both a and b

Fig. 9

Immunostaining with antibodies against RT-97 for labeling axons (a, b) and against myelin basic protein (MBP) for central myelin (c, d) at the lesion site demonstrated the spared tissue. Cross sections from saline-treated (a) and guanosine-treated cords (b) demonstrate a neurofilament (NF) immunoreactivity surrounding the lesion site. c, d Cross sections from saline-treated (c) and guanosine-treated cords reveal myelin at the lesion site. e, f Merger of the two images demonstrates NF (red) and MBP (green) double fluorescent immunolabeling. There were more spared axons and more myelin in the cords around the lesion site from guanosine-treated animals compared to the saline-treated group. Scale bar = 50 μm for all