Research Of Related Mechanisms Of Calpain In Spinal Cord Injury

Serious consequences of spinal cord injury (SCI) include neuronal deathand tissue degeneration leading to neurological dysfunction and even paralysis.The disruption of blood vessels by the primary injury triggers a devastatingsecondary injury process involving multiple cellular and molecularmechanisms of pathogenesis, which ultimately mediates tissue destruction inSCI following the injury. It is well known that there is loss of major myelinproteins such as myelin basic protein (MBP) and proteolipid protein (PLP),cytoskeletal proteins such as microtubule-associated protein 2 (MAP2) andneurofilament protein (NFP), elevation of intracellular free Ca2+ concentrationin the lesion following spinal cord injury in rats. The degradation of myelinand cytoskeletal proteins obviously implicated increased protease activities inSCI. The increased Ca2+ concentration in the SCI lesion does however appearto be the key event in the initiation of a cascade of biochemical reactionsincluding activation of Calpain, a Ca2+-dependent cysteine protease in SCI. Itis now well established that a substantial increase in Calpain activation causedby an increase in intracellular free Ca2+ level following trauma can selectivelydegrade many cytoskeletal and myelin proteins in CNS injuries. The extent ofdegradation of myelin and cytoskeletal proteins reflects an increased Calpainactivity in the SCI lesion. The degradation of various myelin and cytoskeletalproteins concomitant with degeneration of axon and vesiculation of myelinsuggested a crucial role for Calpain in SCI tissue destruction. Sincecytoskeletal and membrane proteins maintain structural integrity of the CNScells, their degradation destabilizes cellular architecture triggering CNS celldeath.A prominent role for Calpain in the pathogenesis of traumatic braininjury was widely accepted. But the related mechanisms of Calpain in ratspinal cord injury have not been well understood yet. In this investigation, wemeasured the activity and transcriptional expression of Calpain, thepathological role of Calpain following SCI in rats, and investigated theneuroprotection effect with Calpain inhibitor E-64-d. It is anticipated thatrationale therapeutic strategies inhibited the activity of Calpain in animalmodels of SCI may eventually lead to an effective treatment of SCI inhumans.Part one Calpain activity and transcriptional expression increased inspinal cord injuryMethods: The models of acute spinal cord injury (Allen's weight dropping)were made. Six Wistar rats were randomly divided into two groups : controlgroup (n=1)and experimental group(n=5).the animals were then sacrificed at 3days after injury. The lesion areas of the spinal cord were cut for study. Theactivity and transcriptional expression of Calpain were examined via Westernand Northren blotting analysis, respectively.Results: The mRNA expression of Calpain was observed in the lesion at 3days following injury, Calpain activity as measured by Calpain-specificdegradation of the endogenous substrate fodrin was found either.Part two Increased Calpain expression is associated with apoptosis inrat spinal cord injury: Calpain inhibitor provides neuroprotectionMethods: The SCI models were induced by dropping a constant weight (10 g)from a height of 10 cm (100 g-cm force) onto an impounder (0.3-cm diameter)gently placed on the spinal cord. 85 Wistar rats were randomly divided into 3groups: sham group (laminectomy performed only, n=5), SCIgroup(n=40),E-64-d group(n=40).Treatment of injured animals beganimmediately after surgery. E-64-d at a dose of 1 mg/kg using 1.5%dimethylsulfoxide (DMSO) as vehicle was administered to 40 SCI rats for 24hours. The other 45 SCI rats received nothing. Animals were sacrificed andthe spinal cords in the lesion were cut at 1,2,3,7,14 day after injury.Immunohistochemical and RT-PCR technique were used to detect theexpressions of Calpain and mRNA level in spinal cord tissue. The level ofneuronal cell was detected by the terminal deoxynucleotidyl transferasemediated DUTP nick end labeling (TUNEL) methods.Results: There was few Calpain expression in normal spinal cord as detectedby immunohistochemical study. The staining index of Calpain increased at1day after injury and peaked at 3d, compared to sham controls. The proteinexpression of Calpain by immunohistochemical staining was similar to theTUNEL time-course date. A positive correlation was seen between Calpainexpression and neural apoptosis(r=0.993). Calpain mRNA increased at 1dayand peaked at 2day after trauma. The correlation between Calpain mRNA andthe level of neural apoptosis was positive(r = 0. 892). Therapeutic effect of theCalpain inhibitor E-64-d was studied. In the E-64-d treated group, the numberof apoptosis cells and the expression of Calpain were decreased as comparedwith those in the SCI group. Statistically analysis showed significantdifference between the SCI group and the treated group.Part three the role of Calpain in the degradation of MAP2 in rat spinalcord injuryMethods: The SCI models were made induced by weight –drop technique. 45wistar rats were randomly divided into 2 groups: control group (laminectomyperformed only, n=5), SCI group(n=40).The expression of Calpain and MAP2were detected by immunohistochemical technique at 1,2,3,7,14 days afterinjury, respectively.Results: The staining index of Calpain detected by immunohistochemicalstudy was increased follwing SCI. However, the staining index of MAP2 wasdecreased in the same time-course. The correlation between Calpain andMAP2 was negative.Part four Activation of CalpainⅠmediate acute reactive gliosis in ratsfollowing spinal cord injuryMethods: We made the SCI models in wistar rats. The animals were randomlyassigned into 6 experimental groups. Treatment of injured animals beganimmediately after surgery. Test solutions were administered by slowly''infusing'' the solution into the lesion site at a rate of 0.1 ml/min using thesurgically implanted Alzet pumps connected with cannula. Rats were given 1ml of either saline containing 1.5% DMSO (n= 10), nifedipine(0.4mM insaline containing 1.5% DMSO, n= 10), Calpain inhibitor I (n=10), or Calpaininhibitor II (1.5 mg/kg in saline containing 1.5% DMSO;n=10).Meanwhile,we took untreated control group(rats which underwent spinal cord injurybut were not treated pharmacologically except for antibiotic therapy, n=6),andsham group(animals that received laminectomy only. n=4) either. Calpaininhibitors I and II were given only one time while all other treatments weregiven three times daily for 2 days. After 2 days of treatment, rats weresacrificed and the spinal cords at the injury site were collected.Immunohistochemical study was used to analysis the immunoreactivity toglial fibrillary acidic protein (GFAP).the number and the average area ofreactive astrocytes was quantified using BioQuant image analysis system.Results: Spinal cord injury resulted in a significant increase in the number ofreactive astrocytes in both gray and white matters. The average size of thereactive astrocytes in the injured spinal cord is much greater than that found innormal tissue. Treatment with nifedipine, and Calpain inhibitor I significantlyreduced both the number and the size of reactive astrocytes in injured spinalcord. Treatment with Calpain inhibitor II was ineffective. Only a few cellsshowed weak GFAP-IR in both gray and white matters of the unoperatedspinal cord. Two days after injury, there was a significant reactive astrogliosisobserved in both gray and white matters of rats with untreated spinal cordinjury or injuried spinal cords treated with vehicle alone. Numerous reactiveastrocytes, characterized by their intense GFAP-IR and hypertrophic cellbodies, accumulated to the injury site. Neither the pattern nor the intensity ofreactive gliosis was significantly different in untreated control rats, ascompared to the response seen in injuried spinal cords treated with vehiclealone. This strongly suggests that the vehicle had no effect on the outcome ofthese experiments. As Calpain inhibitors I and II preferentially inhibit theactivities of μ-Calpain and m-Calpain, respectively, this observations suggestthat the increase in GFAP-IR was mediated mainly by Calpain I.DiscussionCalpain, discovered by Guroff in the CNS, is a disubiquitous intracellularprotease. Research upon Calpain has grown remarkably over the last decadewith identification of many other members of the mammalian Calpain family.Ubiquitous Calpains in mammals exist as two major isoforms, micro-Calpain(μ-Calpain or Calpain 1) and milli-Calpain (m-Calpain or Calpain 2) thatrequire 2–80μM and 0.2–0.8 mM Ca2+ concentrations, respectively, forhalf-maximal activity. Calpain surely participates in numerous intracellularsignaling pathways that are regulated by intracellular free Ca2+ concentrations.The level of Calpain activity is regulated by endogenous activators such asphospholipids and endogenous inhibitors such as calpastatin, and byintracellular Ca2+ concentration. Following traumatic SCI, there is an increasein neutral protease activity at the injury site in rat spinal cord, readilydegrading cytoskeletal proteins and membrane proteins. In the present study,we have assessed the induction of Calpain activity following traumatic SCI bymonitoring the fate of cytoskeletal Calpain substrate, fodrin. Calpain-mediated proteolysis of the a-subunit of fodrin cleaves the subunit at a singlesite. We choose the specific antibodies against this cleaved fragment of fodrin.These antibodies recognize their respective proteolytic fragments only but notthe intact a-subunit of fodrin. Furthermore, these antibodies do not recognizeα-fodrin proteolytic fragments generated by other proteases examined in thatstudy. As such, screening of tissue homogenates using these antibodiesprovides an indirect yet specific assay of Calpain activity. We havedemonstrated that Calpain mRNA transcription and activity is marginallyincreased at 3day compared to sham controls after injury in this study.The results of this investigation indicate that increased Calpain activity inthe lesion has been implicated in the pathogenesis of SCI. MAP2-one ofcytoskeletal proteins, which maintain cellular integrity, are degraded by theactivated Calpain in the lesion of SCI rats in this study. It is now known thatthe secondary injury is responsible for the 'initiation' of apoptosis andprogressive neurodegeneration in SCI. Apoptosis is an active gene-directedcell death process. Thus, therapeutic intervention before initiation ofsecondary injury may protect cells and ameliorate the devastatingconsequences of SCI. In this study, the samples of spinal cord at five timepoint following SCI were investigated in order to assess the extent of Calpainproteinal and mRNA expression in relation to the degree of the cell apoptosisdetected by TUNEL technique. Our data revealed different degrees ofapoptotic cell death after injury following SCI. The finding of a concomitantincrease in Calpain proteinal and mRNA expression in these samples suggestsa role for Calpain in mediation of cell death and tissue destruction.We found the Calpain inhibitor E-64-d to be effective for attenuation ofapoptosis and neurodegeneration in rat SCI. Therapeutic use of Calpaininhibitors may offer some advantages over ion channel blockers and glutamateantagonists, since Calpain mediated proteolysis occurs at a later stage inapoptosis triggered by intracellular free Ca2+ levels and excitotoxicity. Thus, awide window of time provides opportunity for inhibition of increased Calpainactivity and apoptosis of neuronal cells.Astrocytes undergo a process known as reactive gliosis in response to aCNS injury. Among the hallmarks of this process are an increase inimmunoreactivity(IR) to glial fibrillary acidic protein(GFAP), hypertrophy ofastrocytic cell bodies, and proliferation of astrocytes. Reactive astrocytescomprise the most abundant component of the ''glial scar,'' which eventuallyforms within the CNS following injury. It is believed that this glial scar tissueinhibits regeneration in the CNS by forming a physical barrier impeding axoningrowth. By treating rats with Calpain inhibitor I, a Calpain inhibitorpermeable to cells, we significantly reduced reactive gliosis after spinal cordinjury. Calpain inhibitor II, however, while structurally similar to Calpaininhibitor I, failed to prevent reactive gliosis. Since these two Calpaininhibitors preferentially inhibit Calpain I and Calpain II, respectively, ourresults suggest that reactive gliosis following spinal cord injury is mediatedmainly by Calpain I.CONCLUSIONa. Calpain mRNA expression and activity is increased in ratspinal cord injury.b. Increased Calpain expression is associated with necrosis andapoptosis in rat spinal cord injury. Calpain inhibitor E-64-dprovides neuroprotection.c. Activation of Calpain Ⅰmediate acute reactive gliosis ininjured spinal cord in rats.