This study demonstrates a considerable neuroprotective effect of HSYA against spinal cord ischemia/reperfusion injury in rabbits. Animals treated with HSYA exhibited improved neurological scores, reduced tissue destruction and oxidative stress, and decreased neuronal apoptosis.
Reactive oxygen species (ROS) induced damage has been accepted as an important contributor in post-ischemic cell injury and cell death in spinal cords . ROS, consisting of oxygen free radicals and associated entities, are natural products in the metabolism of cells [16, 17]. In pathological conditions, including I/R injuries, ROS are overproduced while antioxidative enzymes are inactivated and antioxidants over-consumed . When the production of ROS overwhelms endogenous antioxidant defenses, excessive ROS would result in the self-perpetuating free radical reaction of lipid peroxidation, which could, subsequently, induce membrane damage, cell lysis, organelle dysfunction, and calcium dyshomeostasis. Since spinal cords are characterized by large lipid content and high oxygenation, they are especially susceptible to lipid peroxidation related cellular damage . Therefore, salvaging the spinal cord from oxidative damage by using antioxidant therapies is an important area of continuing investigation in spinal cord I/R injuries. As a matter of fact, high-dose of methylprednisolone, which is considered the only widely accepted pharmacological therapy currently in use in human spinal cord injuries, confers neuroprotection through its ability to inhibit post-traumatic lipid peroxidation and inflammatory responses.
In traditional Chinese medicine, the flower of the safflower plant, Carthamus tinctorius L., has been extensively used in the treatment of cerebrovascular and cardiovascular diseases. The extracts of Carthamus tinctorius L. contain yellow and red pigments including hydroxysafflor yellow A (HSYA), safflor yellow B, safflomin A, safflomin C, as well as other chemicals. Among all these components, HSYA has been demonstrated as the most active chemicals. In 2003, Zhu et al.  first reported the neuroprotective effects of HSYA on cerebral ischemic injury in both in vivo and in vitro studies. Considering that oxidative stress is an important cause of tissue damage after I/R injury, Jin et al.  examined the possible antioxidative effect of HSYA and demonstrated that HSYA could scavenge hydroxyl radicals and inhibit lipid peroxidation in vitro. The antioxidative effect of HSYA, as proposed by Jin et al., may be attributed to its multiple phenolic hydroxyl groups. In accordance with Jin's study, Tian et al. [20, 21] further demonstrated that HSYA protected the cortex mitochondrial against I/R injuries through scavenging of free radicals, reduction of lipid peroxidation, inhibition of Ca2+ overload, and inhibition of the opening of mitochondrial permeability transition pores (mtPTP). Later, Wei's study of focal cerebral ischemia reperfusion injury showed that, compared with animals that received no pharmacological treatment, animals treated with HSYA revealed reduced MDA content, increased SOD activity and total antioxidative capability in the brain and serum . This study provided direct in vivo evidence that HSYA confers neuroprotection through its antioxidative action. Moreover, as reported by Zhu  and Ye , the neuroprotective effect of HSYA might also be attributed to its inhibition of thrombosis formation and platelet aggregation, its regulation on PGI2/TXA2 (prostaglandin I2/thromboxane A2) ratio and blood rheological changes, as well as its suppression of inflammatory responses.
Although beneficial effects of HSYA have been demonstrated in I/R injuries of various organs, its effect on spinal cord I/R injuries has not been studied yet. Thus, we conducted the present study to investigate the possible neuroprotective effect of HSYA against spinal cord I/R injuries in rabbits. The dose of 10 mg/kg was chosen based on our prior preliminary study and related references which demonstrated remarkable protection of HSYA against brain I/R injury at the dose of 8 mg/kg in rats .
The hypothesis that HSYA could protect against spinal cord I/R injury was accepted because neurological evaluation after reperfusion revealed improved motor function of hinder limbs in animals that received HSYA treatment. However, neurological scores of animals in HSYA group were still lower than that of sham group, suggesting that HSYA alone may not be effective enough to completely restore motor functions. The beneficial effect of HSYA was also confirmed by histopathological study. While severe vacuolization of gray matter and degeneration of motor neurons were observed in the non-treated group, only mild tissue destruction with significant more normal motor neurons were detected in the HSYA-treated group.
To further answer the question of whether the protective effect of HSYA was related to its antioxidative efficiency, we performed the biochemical analysis to study the changes in MDA level and SOD activity in spinal cords. Lipid peroxidation induced by ROS is a primary cause of reperfusion injury in spinal cords when blood flow is restored, and the level of MDA, a relatively stable end product of lipid peroxidation, is considered as indirect evidence of reperfusion injury. In accordance with other researches, a significant increase in MDA content was observed in the spinal cord tissue of rabbits in I/R group . HSYA significantly attenuated the increase of MDA in the tissue. We therefore concluded that the protective effect of HSYA in I/R-induced spinal cord injury may be partly related to its anti-lipid peroxidative properties. SOD, a protective enzyme that scavenges the superoxide radical by catalyzing its dismutation to hydrogen peroxide and oxygen, is the first line of defense against free radical generation. The overproduction of ROS can cause SOD consumption and depletion. In the current study, SOD activity of the spinal cord tissue was greatly decreased by I/R, and was restored by HSYA treatment. However, the precise mechanism of the effect of HSYA on SOD activity remains to be determined.
Remembering that apoptosis is responsible for delayed neuronal cell death after I/R injury , we further studied the effect of HSYA on neuronal apoptosis by TUNEL staining. Compared with control group, HSYA significantly reduced the number of TUNEL-positive cells in the anterior horn of spinal cords. This protective effect of HSYA against neuronal apoptosis may be related to its antioxidative efficiency because the major mechanism of I/R induced apoptosis is attributed to ROS release . Recent studies have revealed that antioxidants attenuated ischemic neuronal apoptosis through Bcl-2 up-regulation and Bax down-regulation . The exact mechanism of HSYA's anti-apoptotic effect will be explored in further studies.