Denise D. Matzelle

Melatonin attenuates calpain upregulation, axonal damage and neuronal death in spinal cord injury in rats

Abstract:  Multiple investigations in vivo have shown that melatonin (MEL) has a neuroprotective effect in the treatment of spinal cord injury (SCI). This study investigates the role of MEL as an intervening agent for ameliorating Ca2+‐mediated events, including activation of calpain, following its administration to rats sustaining experimental SCI. Calpain, a Ca2+‐dependent neutral protease, is known to be involved in the pathogenesis of SCI. Rats were injured using a standard weight‐drop method that induced a moderately severe injury (40 g.cm force) at T10. Sham controls received laminectomy only. Injured animals were given either 45 mg/kg MEL or vehicle at 15 min post‐injury by intraperitoneal injection. At 48 hr post‐injury, spinal cord (SC) samples were collected. Immunofluorescent labelings were used to identify calpain expression in specific cell types, such as neurons, glia, or macrophages. Combination of terminal deoxynucleotidyl transferase (TdT)‐mediated dUTP nick‐end labeling (TUNEL) and double immunofluorescent labelings was used to identify apoptosis in specific cells in the SC. The effect of MEL on axonal damage was also investigated using antibody specific for dephosphorylated neurofilament protein (dNFP). Treatment of SCI animals with MEL attenuated calpain expression, inflammation, axonal damage (dNFP), and neuronal death, indicating that MEL provided neuroprotective effect in SCI. Further, expression and activity of calpain and caspse‐3 were examined by Western blotting. The results indicated a significant decrease in expression and activity of calpain and caspse‐3 in SCI animals after treatment with MEL. Taken together, this study strongly suggested that MEL could be an effective neuroprotective agent for treatment of SCI.

Therapeutic potential of melatonin in traumatic central nervous system injury

Abstract:  A vast literature extolling the benefits of melatonin has accumulated during the past four decades. Melatonin was previously considered of importance to seasonal reproduction and circadian rhythmicity. Currently, it appears to be a versatile anti‐oxidative and anti‐nitrosative agent, a molecule with immunomodulatory actions and profound oncostatic activity, and also to play a role as a potent neuroprotectant. Nowadays, melatonin is sold as a dietary supplement with differential availability as an over‐the‐counter aid in different countries. There is a widespread agreement that melatonin is nontoxic and safe considering its frequent, long‐term usage by humans at both physiological and pharmacological doses with no reported side effects. Endeavors toward a designated drug status for melatonin may be enormously rewarding in clinics for treatment of several forms of neurotrauma where effective pharmacological intervention has not yet been attained. This mini review consolidates the data regarding the efficacy of melatonin as an unique neuroprotective agent in traumatic central nervous system (CNS) injuries. Well‐documented actions of melatonin in combating traumatic CNS damage are compiled from various clinical and experimental studies. Research on traumatic brain injury and ischemia/reperfusion are briefly outlined here as they have been recently reviewed elsewhere, whereas the studies on different animal models of the experimental spinal cord injury have been extensively covered in this mini review for the first time.

The parkinsonian neurotoxin rotenone activates calpain and caspase-3 leading to motoneuron degeneration in spinal cord of Lewis rats

Exposure to environmental toxins increases the risk of neurodegenerative diseases including Parkinsons disease (PD). Rotenone is a neurotoxin that has been used to induce experimental Parkinsonism in rats. We used the rotenone model of experimental Parkinsonism to explore a novel aspect of extra-nigral degeneration, the neurodegeneration of spinal cord (SC), in PD. Rotenone administration to male Lewis rats caused significant neuronal cell death in cervical and lumbar SC as compared with control animals. Dying neurons were motoneurons as identified by double immunofluorescent labeling for terminal deoxynucleotidyl transferase, recombinant-mediated dUTP nick-end labeling-positive (TUNEL(+)) cells and choline acetyltransferase (ChAT)-immunoreactivity. Neuronal death was accompanied by abundant astrogliosis and microgliosis as evidenced from glial fibrillary acidic protein (GFAP)-immunoreactivity and OX-42-immunoreactivity, respectively, implicating an inflammatory component during neurodegeneration in SC. However, the integrity of the white matter in SC was not affected by rotenone administration as evidenced from the non co-localization of any TUNEL(+) cells with GFAP-immunoreactivity and myelin basic protein (MBP)-immunoreactivity, the selective markers for astrocytes and oligodendrocytes, respectively. Increased activities of 76 kD active m-calpain and 17/19 kD active caspase-3 further demonstrated involvement of these enzymes in cell death in SC. The finding of ChAT(+) cell death also suggested degeneration of SC motoneurons in rotenone-induced experimental Parkinsonism. Thus, this is the first report of its kind in which the selective vulnerability of a putative parkinsonian target outside of nigrostriatal system has been tested using an environmental toxin to understand the pathophysiology of PD. Moreover, rotenone-induced degeneration of SC motoneuron in this model of experimental Parkinsonism progressed with upregulation of calpain and caspase-3.

Postinjury estrogen treatment of chronic spinal cord injury improves locomotor function in rats

Spinal cord injury (SCI) causes loss of neurological function and, depending on serverity, may cause paralysis. The only recommended pharmacotherapy for the treatment of SCI is high‐dose methylprednisolone, and its use is controversial. We have previously shown that estrogen treatment attenuated cell death, axonal and myelin damage, calpain and caspase activities, and inflammation in acute SCI. The aim of this study was to examine whether posttreatment of SCI with estrogen would improve locomotor function by protecting cells and axons and reducing inflammation during the chronic phase following injury. Moderately severe injury (40 g · cm force) was induced in male Sprague‐Dawley rats following laminectomy at T10. Three groups of animals were used: sham (laminectomy only), vehicle (dimethyl sulfoxide; DMSO)‐treated injury group, and estrogen‐treated injury group. Animals were treated with 4 mg/kg estrogen at 15 min and 24 hr postnjury, followed by 2 mg/kg estrogen daily for the next 5 days. After treatment, animals were sacrificed at the end of 6 weeks following injury, and 1‐cm segments of spinal cord (lesion, rostral to lesion, and caudal to lesion) were removed for biochemical analyses. Estrogen treatment reduced COX‐2 activity, blocked nuclear factor‐κB translocation, prevented glial reactivity, attenuated neuron death, inhibited activation and activity of calpain and caspase‐3, decreased axonal damage, reduced myelin loss in the lesion and penumbra, and improved locomotor function compared with vehicle‐treated animals. These findings suggest that estrogen may be useful as a promising therapeutic agent for prevention of damage and improvement of locomotor function in chronic SCI.

Cell death in spinal cord injury (SCI) requires de novo protein synthesis. Calpain inhibitor E-64-d provides neuroprotection in SCI lesion and penumbra.

Abstract: Degradation of cytoskeletal proteins by calpain, a Ca2+‐dependent cysteine protease, may promote neuronal apoptosis in the lesion and surrounding areas following spinal cord injury (SCI). Clinically relevant moderate (40 g‐cm force) SCI in rats was induced at T12 by a standardized weight‐drop method. Internucleosomal DNA fragmentation or apoptosis in the lesion was inhibited by 24‐h treatment of SCI rats with cycloheximide (1 mg/kg), indicating a requirement for de novo protein synthesis in this process. To prove an involvement of calpain activity in mediation of apoptosis in SCI, we treated SCI rats with a cell‐permeable calpain inhibitor E‐64‐d (1 mg/kg). Following 24‐h treatment, a 5‐cm‐long spinal cord section centered at the lesion was collected, and divided equally into five segments (1 cm each) to determine calpain activity, as shown by degradation of the 68‐kD neurofilament protein (NFP), and apoptosis as indicated by internucleosomal DNA fragmentation. Neurodegeneration propagated from the site of injury to neighboring rostral and caudal regions. Both calpain activity and apoptosis were readily detectable in the lesion, and moderately so in neighboring areas of untreated SCI rats, whereas these were almost undetectable in E‐64‐d‐treated SCI rats, and absent in sham animals. Results indicate that apoptosis in the SCI lesion and penumbra is prominently associated with calpain activity and is inhibited by the calpain inhibitor E‐64‐d providing neuroprotective benefit.

Calpain inhibitor prevented apoptosis and maintained transcription of proteolipid protein and myelin basic protein genes in rat spinal cord injury

Spinal cord injury (SCI) is associated with progressive neurodegeneration and dysfunction. Multiple cellular and molecular mechanisms are involved in this pathogenesis. In particular, the activation of proteases following trauma can cause apoptosis in the spinal cord. Calpain, a calcium-dependent cysteine protease, plays a major role in apoptosis following trauma. We identified apoptosis and decrease in transcription of the genes for proteolipid protein (PLP) and myelin basic protein (MBP) in five 1-cm long spinal cord segments (S1, distant rostral; S2, near rostral; S3, lesion; S4, near caudal; and S5, distant caudal) 24 h after induction of SCI (40 g.cm force) in rats by weight-drop method. Sham rats underwent laminectomy and did not receive injury. Internucleosomal DNA fragmentation occurred prominently in the lesion (S3), moderately in near segments (S2 and S4), and slightly in distant segments (S1 and S5) of injured rats, indicating the occurrence of apoptosis in the lesion and penumbra. Levels of transcription of PLP and MBP were reduced highly in the lesion and moderately in near segments, suggesting that apoptotic loss of cells impaired biosynthesis of two important structural components of myelin. Immediate administration of the calpain inhibitor E-64-d (1 mg/kg) to injured rats prevented apoptosis and restored transcription of these genes, indicating the therapeutic efficacy of calpain inhibitor for treatment of SCI.

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.

Increased calpain expression is associated with apoptosis in rat spinal cord injury: calpain inhibitor provides neuroprotection.

Calpain content was investigated in the lesion of rat spinal cord at 1, 4, 24, and 72 h following injury induced by the weight-drop (40 g-cm force) technique. Calpain content was increased in the lesion, and was highest at 24 h following injury. μCalpain mRNA level in the lesion was increased by 58.4% (p = 0.0135) at 24 h following trauma, compared to sham. Alterations in mRNA expression in the lesion increased bax/bcl-2 ratio by 20.8% (p = 0.0395) at this time point, indicating a commitment to apoptosis. Therapeutic effect of the calpain inhibitor E-64-d (1 mg/kg) was studied in SCI rats follwing administration for 24 h. Internucleosomal DNA fragmentation (apoptosis) was observed in SCI rats, but not in sham or E-64-d treated rats. These results indicate a new information that E-64-d has the therapeutic potential for inhibiting apoptosis in SCI.

A new mechanism of methylprednisolone and other corticosteroids action demonstrated in vitro: inhibition of a proteinase (calpain) prevents myelin and cytoskeletal protein degradation

The affect of methylprednisolone (MP), an anti-inflammatory drug upon purified calpain and the Ca2+-mediated degradation of endogenous proteins of spinal cord homogenate in vitro has been examined. Activity of calpain purified from rabbit muscle was greatly inhibited in a dose-dependent fashion by MP. A 50% inhibition was obtained with 3.2 mM MP concentration and the activity was inhibited further (80%) at 8.1 mM. More potent inhibition of the purified enzyme (70-80%) was produced by dexamethasone (3.9 mM) and prednisolone (4.1 mM). Calpain-mediated degradation of myelin basic protein (MBP) was also inhibited by MP as was cathepsin B-mediated MBP breakdown. The effect of MP and other steroids upon calcium-mediated degradation of spinal cord homogenate was also evaluated. SDS-PAGE analysis revealed significant inhibition of neurofilament protein breakdown by MP and other corticosteroids. This inhibitory effect was much less than that exerted by the calpain inhibitors calpeptin and/or E64-d. These results indicate that MP acts as a proteinase (calpain) inhibitor and define a new mechanism for its actions.

Inhibition of calpain-mediated apoptosis by E-64 d-reduced immediate early gene (IEG) expression and reactive astrogliosis in the lesion and penumbra following spinal cord injury in rats

Upregulation of calpain, a Ca(2+)-activated cysteine protease, has been implicated in apoptosis and tissue degeneration in spinal cord injury (SCI) that over time spreads from the site of injury to the surrounding regions. We examined calpain content and activity, regulation of immediate early genes (IEGs) such as c-jun and c-fos, reactive astrogliosis as the expression of glial fibrillary acidic protein (GFAP), and apoptosis-related features such as caspase-3 mRNA expression and internucleosomal DNA fragmentation in 1-cm long spinal cord segments (S1, distant rostral; S2, adjacent rostral; S3, lesion or injury; S4, adjacent caudal; and S5, distant caudal) following SCI in rats. Calpain content and production of 150 kD calpain-cleaved alpha-fodrin fragment, expression of IEGs, reactive astrogliosis, and apoptotic features were highly increased in the lesion (S3), moderately in adjacent areas (S2 and S4), and slightly in distant areas (S1 and S5) in SCI rats when compared to sham animals. Administration of the calpain-specific inhibitor E-64-d (1 mg/kg) to SCI rats continuously for 24 h inhibited calpain activity and other factors contributing to apoptosis in the lesion and surrounding areas, indicating that calpain played a key role in the pathophysiology of SCI. The results obtained from this animal model of SCI suggest that calpain inhibitor can provide neuroprotection in patients with SCI.