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 Ca 2+ -mediated events, including activation of calpain, following its administration to rats sustaining experimental SCI. Calpain, a Ca 2+ -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.

Exposure to environmental toxins increases the risk of neurodegenerative diseases including Parkinson's 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.

Parkinson’s disease (PD) is the most common neurodegenerative movement disorder, resulting in dopaminergic (DA) neuronal loss in the substantia nigra pars compacta (SNpc) and damage to extranigral spinal cord neurons. Current therapies do not prevent the disease progression. Hence, developing efficacious therapeutic strategies for treatment of PD is of utmost importance. The goal of this study is to delineate the involvement of calpain-mediated inflammation and neurodegeneration in SN and spinal cord in MPTP-induced parkinsonian mice (C57BL/6N), thereby elucidating potential therapeutic target(s). Increased calpain expression was found localized to tyrosine hydroxylase (TH+) neurons in SN alongside with significantly increased TUNEL positive neurons in SN and spinal cord neurons in MPTP mice. Inflammatory markers Cox-2, caspase-1, and NOS-2 were significantly up-regulated in MPTP mice spinal cord as compared to control. These parameters correlated with the activation of astrocytes, microglia, infiltration of CD4+ / CD8+ T cells and macrophages. We found that subpopulations of CD4+ cells (Th1 & Tregs) were differentially expanded in MPTP mice, which could be regulated by inhibition of calpain with the potent inhibitor calpeptin. Pre-treatment with calpeptin (25 μg/kg, i.p.) attenuated glial activation, T cell infiltration, nigral dopaminergic degeneration in SN, and neuronal death in spinal cord. Importantly, calpeptin ameliorated MPTP-induced altered gait parameters (e.g. reduced stride length and increased stride frequency) as demonstrated by analyses of spatio-temporal gait indices using ventral plane videography. These findings suggest that calpain plays a pivotal role in MPTP-induced nigral and extranigral neurodegenerative processes, and may be a valid therapeutic target in PD.

While multiple molecular mechanisms contribute to midbrain nigrostriatal dopaminergic degeneration in Parkinson’s disease (PD), the mechanism of damage in non-dopaminergic sites within the central nervous system, including the spinal cord, is not well understood. Thus, in order to understand the comprehensive pathophysiology underlying this devastating disease, postmortem spinal cord tissue samples (cervical, thoracic and lumbar segments) from patients with PD were analyzed compared to age-matched normal subjects or Alzheimer’s disease (AD) for selective molecular markers of neurodegeneration and inflammation. Distal axonal degeneration, relative abundance of both sensory and motor neuron death, selective loss of ChAT+ motoneurons, reactive astrogliosis, microgliosis, increased cycloxygenase-2 (Cox-2) expression, and infiltration of T cells were observed in spinal cords of PD patients compared to normal subjects. Biochemical analyses of spinal cord tissues revealed associated inflammatory and proteolytic events (elevated levels of Cox-2, expression and activity of μ- and m-calpain, degradation of axonal neurofilament protein, and concomitantly low levels of endogenous inhibitor – calpastatin) in spinal cords of PD patients. Thus, pathologically upregulated calpain activity in spinal cords of patients with PD may contribute to inflammatory response-mediated neuronal death, leading to motor dysfunction.