Beneficial Effects Of Thymosin β4 And β-aescin On Spinal Cord Injury In The Rat

【Background】Spinal cord injury (SCI) is an issue that has aroused worldwide interest, particularly in recent decades. Severe SCI often leads to lifelong paralysis. Evidence from both clinical and experimental studies indicates that, the pathologic process following SCI is divided into two chronological phases: the primary mechanical injury and a wave of secondary injury triggered by the initial insult The multifaceted secondary injury that results in evolution of the pathological changes are mainly associated with ischemia, edema, immune responses, excitotoxicity, free radical injury, and apoptosis. The spinal cord is highly vascularized to meet its high demand of oxygen and glucose supply, thus ischemia may cause devastating outcomes. Our previous study found that, there were two ischemic zones at the front of the expanding secondary injury, a zone neighboring the lesion where most of the neurons are degenerating or have disappeared, and a farther zone in which most of the neurons appeared relatively normal and considered possible to be rescued. In fact, it has been reported that improving posttraumatic spinal cord blood flow can significantly facilitate functional recovery. That the immune responses considered as a double-edged sword in SCI has been well documented. Although infiltrating immune cells produce neurotrophic factors mediating neuroprotection and regeneration after SCI, it has also been shown that an early inflammatory response and activated macrophages initiated by SCI may participate in neuropathology and loss of motor function. In those secondary damaging processes after SCI, both neurons and oligodendrocytes are vulnerable to various harmful factors. Due to the loss of oligodendrocytes and subsequent axonal demyelination of surviving axons and impair their conductive capacity, it seems reasonable to expect that reducing oligodendrocyte death and improving axonal remyelination holds potential for the treatment of SCI.Tβ4, a highly conserved 4.9 kD polypeptide, comprises of 43 amino acids, which was first isolated from bovine thymus. It is a natural polypeptide that is ubiquitously distributed in almost all cells with relatively higher levels in platelets and white blood cells. The angiogenesis, anti-inflammation, wound repair and cell survival properties of Tβ4 are highly relevant to the SCI. We have recently formulated an idea of early neurosurgery of spinal cord contusion with great success. The patients received only surgery and rehabilitation. No medication that might improve recovery was given to avoid confusion of credit of the surgery. Therefore, search for effective drugs that may fill in the big space left for improving the outcome of operation may contribute substantially to the treatment of spinal cord contusion. The present study was focused on its angiogenic, neuronal protective and anti-inflammatory effects and the results showed that these three aspects of Tβ4 worked well in reducing the secondary injury and improve locomotion.【Objective】The present study was aimed to investigate the therapeutic value for SCI of its angiogenic, neuroprotective and anti-inflammatory properties. We used a rat SCI model of bilateral compression of the spinal cord.【Methods】The SD rats were randomly divided into 3 groups: sham (n = 5), Tβ4 (n = 15), and normal saline (n = 15). After anesthesia, a rat SCI model of lateral compression of the spinal cord was produced using a pair of modified forceps. Tβ4 or normal saline was administrated i.p. 30 min after injury, and 2 doses on the first day of every other 3 days. Histological tests were performed to assess blood vessels, neuronal survival, and lesion size. Western blot assay was executed to detect the expression of specifical marker ED1 for activated microglia and infiltrated macrophages and specifical marker MBP for mature oligodendrocytes. All rats were allowed to surviv for 7 days. All rats underwent behavioral assessments at every time point.【Results】Our data demonstrated that the size of lesion area delineated by GFAP was decreased by 32.1% in the Tβ4 group compared with the saline group. The changes of total length of the blood vessels and total number of neurons within the gray matter occurred in the areas 1 mm rostral and caudal to the lesion edge. The density of total length of blood vessels in the rostral 1 mm was 14.72±0.17 mm/mm~2 in the Tβ4 treated group versus 12.97±0.45 mm/mm~2 in the saline group, in the caudal 1 mm was 13.35±0.13 mm/mm~2 versus 11.40±0.39 mm/mm~2, respectively. The number of neurons in the Tβ4 treated group in the rostral and caudal 1 mm zones were 335.1±11.1/mm~2 and 317.7±9.3 /mm~2, respectively, whereas in the saline group the rostral and caudal zones hosted far less NeuN-immunoreactive cells, 284.8±15.4/mm~2 and 262.1±16.6 /mm~2, respectively. Furthermore, there was a close correlation between the effects on blood vessel length and neuronal survival. Western blotting assay showed that, the ED1 expression in the Tβ4 group was 35.9% less than that in the saline group; the MBP level was only decreased by 20.1% in the Tβ4 group but clearly decreased by 44.3% in the saline group compared with normal control. Locomotor recovery was tested using the BBB score and footprint analysis. All of the behavioral assessments were markedly improved.【Conclusions】Here, for the first time, our present study shows that, systemic administration of Tβ4 after acute SCI in rats, remarkably reduces the lesion size and some pathological changes of the injured spinal cord, and accelerates locomotion recovery, most probably, by stimulating angiogenesis, increasing neuronal survival, reducing inflammatory infiltration and demyelination. Given its known safety in clinical uses and the beneficial effects on spinal cord injury, Tβ4 can well be a good candidate for treating spinal cord contusion.Part II: Beneficial effects ofβ-aescin on spinal cord injury in the rat【Background】The severity of neurological disturbance, induced by traumatic SCI, is not only associated with the degree of the mechanical insult to the tissue, but also closely correlated to progressive tissue loss beyond the primary injury site. Multiple secondary damaging factors are involved in the latter process, including ischemia, immune response, glutamate excitotoxicity, free radical generation, reactive oxygen species, lipid peroxidation (LPO), and apoptosis, to name a few. LPO is a self-perpetuating form of free radical damage that is believed to be a major factor in the spread of deterioration after SCI to regions of spared tissue outside the original zone of trauma. Malondialdehyde (MDA), the principal end product of polyunsaturated fatty acid peroxidation in cell membranes, considered as a biological marker of LPO, is a highly toxic molecule, aggravating the damage beyond membrane lipids by interacting with amino acids, proteins and DNA. The immune system likely plays a critical role in the secondary degenerative process. An intense local inflammatory response, typed by activation of resident microglia and influx of hematogenous neutrophils and macrophages, is rapidly triggered by traumatic SCI. Those cells produce free radicals including superoxide anion and nitric oxide during these processes inducing apoptosis in neurons and glia via the irreversible oxidation of proteins, lipids and nucleic acids. The LPO and inflammatory responses are therefore good targets for the exploration of pharmacological strategies to treat acute SCI. At present, the methylprednisolone having the properties of anti-peroxidation and anti-inflammation remains the only neuroprotective agent in clinic pharmacotherapy for acute SCI, but its use existing extensive controversies due to undesirable side effects. Thus we are interested in seeking for a novel therapeutic approach for acute phase SCI without significant side effects.Aescin, the major active principle from aesculus hippocastanum, was subjected to a natural mixture of triterpene saponins. It has two isomers ofαandβ. Among which,β-aescin shown numerous pharmacological properties of anti-edema, anti-inflammation, anti-oxidation, increasing venous tone, and protecting vascular endothelial cells.β-aescin appears to be the active component of the mixture and is the molecular form present in major available pharmaceutical products. It has been widely applied in haemorrhoids, chronic venous insuf?ciency, peripheral vascular diseases, and postoperative, postburn or posttraumatic edema in clinical practices with very few adverse effects. The aim of this study was to investigate the possible effects ofβ-aescin on LPO and cellular in?ammation after traumatic SCI when the agent was administered intraperitoneally. We also determined whetherβ-aescin could improve functional deficits.【objective】To investigate the protective effects ofβ-aescin on acute traumatic spinal cord injury (SCI) in rats【Methods】We examined this hypothesis in a rat model of moderate spinal cord contusion. All rats were assigned into 3 groups: 1. Sham-operated control group (n = 10): only received a laminectomy with no additional treatment; 2.β-aescin group (n = 18): SCI plus 1 mlβ-aescin.i.p; 3. Saline control group (n = 18): SCI plus 1 ml saline.i.p. 30 min after injury, the spinal cord-injured rats were treated withβ-aescin (1.0 mg/kg bw i.p.) or normal saline, and received additional dose t.i.d for 3 consecutive days. Locomotor deficits induced by SCI were assessed using the BBB score and footprint analysis. Biochemical assays were executed to detect the level of malondialdehyde (MDA) and the activity of myeloperoxydase (MPO). Histological tests were performed to analyze the lesion size, and activated microglia and recruited macrophages.【Results】Our data indicated that all of the behavioral assessments were markedly improved. In addition,β-aescin treatment significantly reduced MDA level by 67% and MPO activity by 50% at 24 hr after injury, decreased lesion cavitation by 29.3% and ED1 positive area by 32.3% at 14 days after injury.【Conclision】The present study showed thatβ-aescin treatment strikingly reduced lipid peroxydation (LPO), suppressed microglia activation and hematogenous leukocyte infiltration, and facilitated reparation after SCI.