ABSTRACT
Spinal cord injury (SCI) is a devastating neurologic injury, and currently, the only recommended pharmacotherapy is high-dose methylprednisolone, which has limited efficacy. Estrogen is a multi-active steroid with anti-oxidant and anti-apoptotic effects. Estrogen may modulate intracellular Ca2+ and prevent inflammation. For this study, male rats were divided into three groups. Sham-group animals received a laminectomy at T12. Injured rats received both laminectomy and 40 gram centimeter force SCI. Estrogen-group rats received 4 mg/kg 17beta-estradiol (estrogen) at 15 min and 24 hr post-injury, and vehicle-group rats received equal volumes of dimethyl sulfoxide. Animals were sacrificed at 48 hr post-injury, and 1-cm segments of the lesion, rostral penumbra, and caudal penumbra were excised. The degradation of 68 kD neurofilament protein (NFP) and estrogen receptors (ER) was examined by Western blot analysis. Protein levels of calpain and the activities of calpain and caspase-3 were also examined. Levels of cytochrome c were determined in both cytosolic and mitochondrial fractions. Cell death with DNA fragmentation was examined using the TUNEL assay. At the lesion, samples from both vehicle and estrogen treated animals showed increased levels of 68 kD NFP degradation, calpain content, calpain activity, cytochrome c release, and degradation of ERalpha and ERbeta, as compared to sham. In the caudal penumbra, estrogen treatment significantly attenuated 68 kD NFP degradation, calpain content, calpain activity, levels of cytosolic cytochrome c, and ERbeta degradation. At the lesion, vehicle-treated animals displayed more TUNEL+ cells, and estrogen treatment significantly attenuated this cell death marker. We conclude that estrogen may inhibit cell death in SCI through calpain inhibition.
Subject(s)
Apoptosis/drug effects , Calpain/metabolism , Estrogens/therapeutic use , Spinal Cord Injuries/drug therapy , Analysis of Variance , Animals , Blotting, Western/methods , Boron Compounds/metabolism , Caspase 3/metabolism , Cytochromes c/metabolism , Disease Models, Animal , Enzyme Activation/drug effects , Fluorescent Antibody Technique/methods , In Situ Nick-End Labeling/methods , Laminectomy/methods , Male , Neurofilament Proteins/metabolism , Rats , Rats, Sprague-Dawley , Receptors, Estrogen/metabolism , Time FactorsABSTRACT
Spinal cord injury (SCI) is a devastating neurologic injury with functional deficits for which the only currently recommended pharmacotherapy is high-dose methylprednisolone, which has limited efficacy. Estrogen is a multi-active steroid that has shown antiinflammatory and antioxidant effects, and estrogen may modulate intracellular Ca(2+) and attenuate apoptosis. For this study, male rats were divided into three groups. Sham group animals received a laminectomy at T12. Injured rats received both laminectomy and 40 g x cm force SCI. Estrogen-group rats received 4 mg/kg 17beta-estradiol (estrogen) at 15 min and 24 hr post-injury, and vehicle-group rats received equal volumes of dimethyl sulfoxide (vehicle). Animals were sacrificed at 48 hr post-injury, and 1-cm-long segments of the lesion, rostral penumbra, and caudal penumbra were excised. Inflammation was assessed by examining tissue edema, infiltration of macrophages/microglia, and levels of cytosolic and nuclear NFkappaB and inhibitor of kappa B (IkappaBalpha). Myelin integrity was examined using Luxol fast blue staining. When compared to sham, vehicle-treated animals revealed increased tissue edema, increased infiltration of inflammatory cells, decreased cytosolic levels of NFkappaB and IkappaBalpha, increased levels of nuclear NFkappaB, and increased myelin loss. Treatment of SCI rats with estrogen reduced edema and decreased inflammation and myelin loss in the lesion and penumbral areas, suggesting its potential as a therapeutic agent. Further work needs to be done, however, to elucidate the neuroprotective mechanism of estrogen.
Subject(s)
Estrogens/pharmacology , Myelitis/drug therapy , Nerve Degeneration/drug therapy , Spinal Cord Injuries/drug therapy , Spinal Cord/drug effects , Animals , Biomarkers/metabolism , Chemotaxis, Leukocyte/drug effects , Chemotaxis, Leukocyte/physiology , Disease Models, Animal , Edema/drug therapy , Edema/physiopathology , Edema/prevention & control , Estrogens/therapeutic use , Gliosis/drug therapy , Gliosis/physiopathology , Gliosis/prevention & control , I-kappa B Proteins/metabolism , Inflammation Mediators/metabolism , Male , Microglia/drug effects , Myelin Sheath/drug effects , Myelin Sheath/metabolism , Myelitis/metabolism , Myelitis/physiopathology , NF-kappa B/metabolism , Nerve Degeneration/physiopathology , Nerve Degeneration/prevention & control , Neuroprotective Agents/pharmacology , Neuroprotective Agents/therapeutic use , Rats , Rats, Sprague-Dawley , Spinal Cord/metabolism , Spinal Cord/physiopathology , Spinal Cord Injuries/metabolism , Spinal Cord Injuries/physiopathology , Treatment OutcomeABSTRACT
While the average age for patients admitted with spinal cord injury is 32 years, patients under the age of 16 account for 5% of spinal cord injured persons. For these younger patients, an increased mortality up to 24 h post-injury has been reported, however, survivors may regain more function than their adult counterparts, suggesting that age may play a role in injury tolerance. While the use of growth factors as a therapy for spinal cord injury is well researched, the response of the developing cord to secondary injury has not been thoroughly investigated. Following spinal cord injury, Ca(2+) influx can activate enzymes such as calpain, a Ca(2+)-dependent protease, which plays a role in the pathogenesis of spinal cord injury in rats. The present investigation revealed that following spinal cord injury, calpain upregulation was significantly less (15.3%) in the 21-day-old rats than in either 45-day-old (70%) or 90-day-old (99.6%) rats, as shown by Western blot and in situ immunofluorescent studies. Expression of the endogenous calpain inhibitor, calpastatin, was significantly higher in juvenile rats than adult rats. Juvenile rats with spinal cord injury also showed a reduced Bax:Bcl-2 ratio (4:1 vs. 6:1), reduced caspase-3 staining, reduced myelin loss (3% vs. 18%), and less neuronal DNA damage, as compared to older rats. These results suggest that increased calpastatin levels found in juvenile rats muted calpain activity and neuronal apoptosis, following spinal cord injury.
Subject(s)
Apoptosis/physiology , Calcium-Binding Proteins/biosynthesis , Calpain/physiology , Neurons/enzymology , Spinal Cord Injuries/enzymology , Age Factors , Animals , Calcium-Binding Proteins/physiology , Female , Hydrolysis , Rats , Rats, Sprague-DawleyABSTRACT
The following review is a brief discussion about spinal cord injury and the possibility of using estrogen as a neuroprotective agent. There are several pathways by which secondary cell death can occur following spinal cord injury, including infiltration of inflammatory cells, generation of reactive oxygen species, decreases in spinal cord blood flow, and increases in intracellular Ca(2+) levels. This secondary damage leads to apoptotic cell death, and the neuroprotective effects of pharmacologic agents have been investigated using experimentally induced spinal cord injury in animals. Currently, only high-dose methylprednisolone is advocated for the treatment of patients following spinal cord injury. Estrogen has been shown to be neuroprotective in both in vitro and in vivo studies. There are several possible mechanisms of action by which estrogen may attenuate damage following spinal cord injury and improve functional outcome. Estrogen has been shown to have anti-inflammatory properties. Estrogen levels are correlated with an increase in post-traumatic blood flow to injured tissue. Estrogen may also upregulate protein levels of anti-apoptotic Bcl-2 and may attenuate the post-traumatic influx of Ca(2+).
