Recombinant human erythropoietin counteracts secondary injury and markedly enhances neurological recovery from experimental spinal cord trauma

Abstract

Erythropoietin (EPO) functions as a tissue-protective cytokine in addition to its crucial hormonal role in red cell production. In the brain, for example, EPO and its receptor are locally produced, are modulated by metabolic stressors, and provide neuroprotective and antiinflammatory functions. We have previously shown that recombinant human EPO (rhEPO) administered within the systemic circulation enters the brain and is neuroprotective. At present, it is unknown whether rhEPO can also improve recovery after traumatic injury of the spinal cord. To evaluate whether rhEPO improves functional outcome if administered after cord injury, two rodent models were evaluated. First, a moderate compression of 0.6 N was produced by application of an aneurysm clip at level T3 for 1 min. RhEPO (1,000 units per kg of body weight i.p.) administered immediately after release of compression was associated with partial recovery of motor function within 12 h after injury, which was nearly complete by 28 days. In contrast, saline-treated animals exhibited only poor recovery. In the second model used, rhEPO administration (5,000 units per kg of body weight i.p. given once 1 h after injury) also produced a superior recovery of function compared with saline-treated controls after a contusion of 1 N at level T9. In this model of more severe spinal cord injury, secondary inflammation was also markedly attenuated by rhEPO administration and associated with reduced cavitation within the cord. These observations suggest that rhEPO provides early recovery of function, especially after spinal cord compression, as well as longer-latency neuroprotective, antiinflammatory and antiapoptotic functions.

Figure 1

(A) Neurological (motor) scores of animals assessed over 72 h after removal of aneurysm clip in open field testing show significant improvement earlier for animals receiving either a single (1,000 units/kg bw i.p.) or 3 doses of rhEPO (3,000 units/kg of bw total; n = 14 each group). (B) Neurological scores of animals assessed for 1 month after removal of an aneursym clip show that animals that received rhEPO exhibited nearly normal motor function. In contrast, saline-treated animals regained much less function (n = 7 each group; *, P < 0.05; **, P < 0 01, ***, P < 0.001).

Figure 2

A. Motor scores of animals assessed for 1 week show that rhEPO given as 5,000 units/kg of bw i.p. once was as effective as 7 doses of rhEPO. Significant recovery of neurological function was observed by 4 days in the rhEPO groups. A dose of 500 units/kg of bw was intermediate in effectiveness. (B) Recovery of neurological function steadily continued over 28 days for animals given a single dose of rhEPO (5,000 units/kg of bw i.p.), such that nearly full recovery was obtained. In contrast, saline-treated animals did not materially improve after 14 days following injury. (n = 6 each group; **, P < 0.01; ***, P < 0.001).

Figure 3

Motor function as assessed by swimming demonstrated improvement by 4 days after injury for rhEPO at high dose (5,000 units/kg of bw i.p. × 7 days). In contrast, a lower dose of rhEPO (500 units/kg of bw i.p.) was no more effective than saline. (n = 12 each group; **, P < 0.01).

Figure 4

Representative hemotoxylin-and-eosin-stained rat spinal cord sections obtained at the lesion epicenter 7 days after injury. (Lower) Saline-treated. (Upper) rhEPO-treated (5,000 units/kg bw i.p. daily for 7 days). Extensive white matter preservation can be observed in the rhEPO-treated group. In contrast, cavitation (asterisks) occupied the central cord of saline-treated animals, surrounded by swollen and fragmented axons. Numerous inflammatory cells were also present throughout (not seen at this magnification). Cavitation volume was reduced in the rhEPO-treated animals by ≈25%. Many TUNEL-positive nuclei corresponding to oligodendrocytes were observed in the fasciculus cuneatus only in the saline-treated animals (see text).