Estrogen attenuated markers of inflammation and decreased lesion volume in acute spinal cord injury in rats.

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.

Upregulation of calpain correlates with increased neurodegeneration in acute experimental auto-immune encephalomyelitis.

Although calpain up-regulation is well established in experimental auto-immune encephalomyelitis (EAE), a link between increased calpain expression and activity and neurodegeneration has not been examined. Therefore, spinal cord tissue from Lewis rats with EAE was examined to test the hypothesis that increased calpain expression in neurons would correlate with increased cell death and axonal damage in a time-dependent manner following EAE induction. We found that increased calpain expression in EAE corresponded to increased TUNEL-positive neurons and to increased expression of dephosphorylated neurofilament protein, markers of cell death and axonal degeneration, respectively. An increase in internucleosomal DNA fragmentation in EAE spinal cord suggested that cell death was, at least partially, due to apoptosis. Axonal damage was further demonstrated in EAE spinal cord compared with control via morphological analysis, revealing granular degeneration of filament and microtubule integrity, loss of myelin, and mitochondrial damage. Calcium (Ca2+) influx, which is required for calpain activation, was also increased in EAE spinal cord. From these findings, we conclude that increases in Ca2+-induced calpain activity may play a crucial role in neurodegeneration in acute EAE.

Higher calpastatin levels correlate with resistance to calpain-mediated proteolysis and neuronal apoptosis in juvenile rats after spinal cord injury.

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.

Early induction of secondary injury factors causing activation of calpain and mitochondria-mediated neuronal apoptosis following spinal cord injury in rats.

To investigate a potential relationship between calpain and mitochondrial damage in spinal cord injury (SCI), a 40 gram-centimeter force (g-cm) injury was induced in rats by a weight-drop method and allowed to progress for 4 hr. One-centimeter segments of spinal cord tissue representing the adjacent rostral, lesion, and adjacent caudal areas were then removed for various analyses. Calcium green 2-AM staining of the lesion and penumbra sections showed an increase in intracellular free calcium (Ca(2+)) levels following injury, compared with corresponding tissue sections from sham-operated (control) animals. Western blot analysis showed increased calpain expression and activity in the lesion and penumbra segments following SCI. Double-immunofluorescent labeling indicated that increased calpain expression occurred in neurons in injured segments. Western blot analysis also showed an increased Bax:Bcl-2 ratio, indicating the induction of the mitochondria-mediated cell death pathway in the lesion and penumbra. The morphology of mitochondria was altered in lesion and penumbra following SCI: mostly hydropic change (swelling) in the lesion, with the penumbra shrunken or normal. At 4 hr after induction of injury, a substantial amount of cytochrome c had been released into the cytoplasm, suggesting a trigger for apoptosis through caspase 3 activation. Neuronal death after 4 hr of injury was detected by a combined TUNEL and double-immunofluoresence assay in the lesion and penumbra sections of injured cord, compared with sham controls. These results suggest that an early induction of secondary factors is involved in the pathogenesis of SCI. The increased Ca(2+) levels could activate calpain and mediate mitochondrial damage leading to neuronal death in lesion and penumbra following injury. Thus, secondary injury processes mediating cell death are induced as early as 4 hr after the injury, and calpain and caspase inhibitors may provide neuroprotection.

Calpain and calpastatin expression in primary oligodendrocyte culture: preferential localization of membrane calpain in cell processes.

The cellular localization of calpain is important in understanding the roles that calpain may play in physiological function. We, therefore, examined calpain expression, activity, and immunofluorescent localization in primary cultures of rat oligodendrocytes. The mRNA expression of m-calpain was 64.8% (P = 0.0033) and 50.5% (P = 0.0254) higher than that of mu-calpain and calpastatin, respectively, in primary culture oligodendrocytes. The levels of mRNA expression of mu-calpain and calpastatin were not significantly different. As revealed by Western blotting, cultured oligodendrocytes contained a 70 kD major band identified by membrane m-calpain antibody, a 80 kD band recognized by cytosolic m-calpain antibody, and calpastatin bands ranging from 45 to 100 kD detected by a calpastatin antibody. Calpain activity in oligodendrocytes was determined by Ca(2+)-dependent 71.2% degradation of endogenous myelin basic protein compared with control; this activity was inhibited significantly (P = 0.0111) by EGTA and also substantially by calpeptin. Localization of calpain in cultured oligodendrocytes revealed strong membrane m-calpain immunofluorescence in the oligodendrocyte cell body and its processes. In contrast, the cytosolic antibody stained primarily the oligodendrocyte cell body, whereas the processes were stained very weakly or not at all. These results indicate that the major form of calpain in glial cells is myelin (membrane) m-calpain. The dissimilar localization of cytosolic and membrane m-calpain may indicate that each isoform has a unique role in oligodendrocyte function.

Molecular evidence of apoptotic death in malignant brain tumors including glioblastoma multiforme: upregulation of calpain and caspase-3.

Cell death in the core of human brain tumors is triggered by hypoxia and lack of nutrients, but the mode of cell death whether necrosis or apoptosis is not clearly defined. To identify the role of apoptosis in brain tumor cell death, we investigated macromolecular (RNA and protein) synthesis and activity in the central to peripheral region of benign [desmoplastic infantile ganglioglioma (DIG) and transitional meningioma (TMG)] and malignant [ependymoma (END), anaplastic astrocytoma (APA), and glioblastoma multiforme (GBM)] brain tumors derived from five patients who had not received previously radiotherapy or chemotherapy. Normal brain tissue (NBT) served as control. RT-PCR analysis of tumor tissues covering central to peripheral regions detected mRNA overexpression of pro-apoptotic gene bax in malignant tumors, indicating a commitment to apoptosis. The mRNA expression of calpain (a Ca(2+)-dependent cysteine protease) and calpastatin (endogenous calpain inhibitor) was altered resulting in an elevated calpain/calpastatin ratio. Calpain content and activity were increased, suggesting a role for calpain in cell death. In the mitochondria-dependent death pathway, caspase-9 and caspase-3 were also overexpressed in tumors. The increased caspase-3 activity cleaved poly(ADP-ribose) polymerase (PARP). Agarose gel electrophoresis detected a mixture of random and internucleosomal DNA fragmentation in malignant brain tumors. Overexpression of pro-apoptotic bax, upregulation of calpain and caspase-3, and occurrence of internucleosomal DNA fragmentation are now presented indicating that one mechanism of cell death in malignant brain tumors is apoptosis, and that enhancement of this process therapeutically may promote decreased tumor growth.