calpeptin and methylprednisolone inhibit apoptosis in rat spinal cord injury

261

Calpeptin and Methylprednisolone Inhibit Apoptosis in Rat Spinal Cord Injury

SWAPAN K. RAY, GLORIA G. WILFORD, DENISE C. MATZELLE, EDWARD L. HOGAN, AND NAREN L. BANIKa

Department of Neurology, Medical University of South Carolina, Charleston,South Carolina 29425, USA

ABSTRACT: Intracellular free Ca2� and free radicals are increased followingspinal cord injury (SCI). These can activate calpain to degrade cytoskeletalproteins leading to apoptotic and necrotic cell death. Primary injury triggers acascade of secondary injury, which spreads to rostral and caudal areas. Wetested calpain involvement in apoptosis in five 1-cm segments of rat spinal cordwith injury (40 g-cm) induced at T12 by weight-drop. Animals were immedi-ately treated with calpeptin (250 �g/kg) and methylprednisolone (165 mg/kg)and sacrificed at 48 hr. Untreated SCI rats manifested 68-kD neurofilamentprotein (NFP) degradation (indicating calpain activity), and internucleosomalDNA fragmentation (indicating apoptosis). Both calpain activity and apoptosiswere highest in the lesion, and decreased with increasing distance from the le-sion. Treatment decreased 68-kD NFP degradation with reduction in apoptosisin all five areas. Thus, calpeptin and methylprednisolone are found to be neu-roprotective in SCI.

INTRODUCTION

The rat model of spinal cord injury (SCI) is useful to examine a role for calpain,the Ca2+-dependent cysteine protease, in causing cytoskeletal protein degradationand programmed cell death (PCD) in the central nervous system (CNS). Apoptosisor PCD is mediated by activation of several cysteine proteases in response to a vari-ety of factors including intracellular free Ca2+ and free radicals. The levels of thesePCD-promoting factors are increased in SCI. One of the most important events inapoptosis is the activation of cysteine proteases.1 Apoptotic death of neural cells, es-pecially neurons and oligodendrocytes, in the spinal cord after trauma will disruptand destroy the axon-myelin structural unit and, therefore, impair impulse conduc-tion. Apoptosis has also been implicated in ischemia and in retinal ganglion cells af-ter optic nerve axotomy.2,3

Since cytoskeletal and membrane proteins are degraded due to activation ofcalpain, many investigators have been interested in finding a role for calpain in apo-ptosis.4 The level of intracellular free Ca2+ is increased during injury, causing acti-vation of Ca2+-dependent proteases and eventually apoptotic cell death.5,6 It has

aCorresponding author: Naren L. Banik, Ph.D., Department of Neurology, Medical Universityof South Carolina, 171 Ashley Avenue, Charleston, SC 29425. Phone, 843/792-3946; fax, 843/792-8626.

e-mail, [email protected]

262 ANNALS NEW YORK ACADEMY OF SCIENCES

been reported that intracellular free Ca2+ is greatly increased in experimental spinalcord trauma.7 Intracellular free Ca2+ is absolutely required for activation of calpain,which may therefore be one of the cysteine proteases responsible for mediation ofcell death in SCI. Cytoskeletal proteins, which maintain cellular integrity, are de-graded by calpain activity during apoptosis.8,9 A role for calpain in apoptosis of neu-rons has been reported.10 Recently we have reported the involvement of calpain inmediation of apoptosis in glial cells.11 Several enzymes including lipases and pro-teases are activated by Ca2+. The activation of Ca2+-dependent phospholipase A2(PLA2) causes neurotoxicity by catalyzing release of arachidonic acid from mem-brane phospholipids followed by production of free radicals. Moreover, activatedPLA2 alters membranes facilitating influx of Ca2+ as well as release of Ca2+ fromthe internal stores.12 Infiltration and activation of a variety of inflammatory cells inthe CNS during the acute phase of SCI releases numerous inflammatory mediatorsand free radicals. Among proteases, calpain is activated in response to elevated in-tracellular free Ca2+ levels.13,14 Activated calpain can induce proteolytic modifica-tions in a number of proteins associated with multiple signaling cascades formediating neural cell death.

Two major calpain isoforms are µcalpain and mcalpain, which are activated byµM and mM Ca2+ concentration, respectively. Each isoform consists of a 30-kD reg-ulatory subunit and an 80-kD catalytic subunit.15 The regulatory subunits are iden-tical in both calpain isoforms, but the catalytic subunits are different. The regulatorysubunit originates from one gene, and the catalytic subunits from different genes.16

Calpastatin is an endogenous protein inhibitor, which regulates the activity of bothcalpain isoforms,17 and the level and efficiency of calpastatin could play a criticalrole in preventing calpain-mediated proteolysis.18 However, it should be noted thatthe level of calpastatin is decreased by calpain-mediated proteolysis.19 It is alsonoteworthy that calpains exist in the cells as inactive proenzymes, which are activat-ed by an increase in Ca2+ levels.

SOURCE OF CALPAIN IN SCI

The primary injury to the cord causes several changes, including disruption ofblood vessels and alteration of membrane integrity, that play important roles in ini-tiating the devastating secondary neuropathophysiological changes. Since degener-ating of myelinated axons and degradation of proteins occur before infiltration ofimmune cells, it is likely that the early increase of calpain activity is derived fromendogenous neural cells (neurons, oligodendrocytes, astrocytes, and microglia),while later there is a contribution from infiltrating inflammatory cells (lymphocytesand mononuclear phagocytes). Increased calpain levels have been found in macroph-ages, astrocytes, microglia, neurons and myelin in SCI.20 Any increase in calpain ex-pression and activity early in the injury is likely due to elevated levels of intracellularfree Ca2+, inflammatory cytokines, and arachidonic acid. Later, an increase incalpain activity in SCI may derive from reactive astrocytes and infiltrating immunecells. The current evidence indicates that arachidonic acid, its metabolites, and cy-tokines all accumulate in CNS injury.21–23

263RAY et al.: PREVENTION OF APOPTOSIS IN SCI

FREE Ca2� AND FREE RADICALS IN SCI

It is now thought that Ca2+ influx in SCI results from cell damage in the lesion,and alteration in cell permeability in the penumbra.7 An increase in intracellular freeCa2+ and free radicals causes cell death in CNS injury, ischemia, stroke, andglutamate neurotoxicity.24 Free Ca2+ is also released from intracellular organellessuch as mitochondria and microsomes.25 Free radicals and other inflammatory me-diators are generated during conversion of arachidonic acid to eicosanoids followingactivation of inflammatory cells,26,27 which are infiltrated in the acute phase of SCIin animal models.28–30 Lipid inflammatory mediators such as eicosanoids, prostag-landins, thromboxane, and leukotrienes accumulate following infiltration of inflam-matory cells. Kininogen (the precursor of kinins) and kinins also progressivelyaccumulate in SCI. Kinins activate phospholipases, which in turn release arachidon-ic acid and produce free radicals.31 In addition to intracellular free Ca2+, other fac-tors including such kinins and free radicals can stimulate calpain activity in themediation of cell death.

A ROLE FOR CALPAIN IN APOPTOSIS IN SCI

The primary injury to the cord causes cell membrane disruption, axon-myelin dis-integration, and microvessel destruction.32 The secondary injuries, initiated by anumber of factors following primary injury, can cause cell death by necrosis or/andapoptosis. Necrosis is irreversible, and marked by damage to the plasma membranceand leakage of cell constituents in to extracellular space fluid. Apoptosis or PCD ispreventable, as it is activated by a number of factors including Ca2+ and free radi-cals.33 Cells undergoing apoptosis are cleared by proteolytic digestion.34 There hasbeen a long-held assumption that necrosis may be the mode of cell death in the SCIlesion,28 and recent implication of excitotoxicity in the pathogenesis of SCI is con-sistent with this view.35,36 Excitotoxicity is characterized by neuronal cell swelling,which is documented in the necrosis of cortical neurons in culture.37 Although thedeath of neurons and glial cells in the lesion after SCI is likely to be necrotic, it maybe largely apoptotic in the penumbra and adjacent areas. Apoptotic death is regulatedby several factors initiating an intrinsic suicide program in the cells. The occurrenceof apoptosis is an important part of secondary injuries in SCI, and has recently beenreported by several laboratories besides ours.38–40

Since calpain expression and activity are increased in the SCI lesion,41,42 calpainmay be one of the proteases responsible for mediating apoptotic cell death after SCI.In response to increased intracellular Ca2+ levels, calpains are activated by autolyticcleavage of N-terminal peptides, and this limited autolysis is considered to be thekey mechanistic step in the activation of calpains.43 Structural proteins and other mi-crotubular proteins have been shown to be partially degraded by calpain.15,44 To thisend, we have investigated the degradation of 68-kD neurofilament protein (NFP) inthe lesion and adjacent areas of spinal cord in a rat model of SCI (FIG. 1). The lossof 68-kD NFP is found to be highest at the site of lesion, and decreases in neighbor-ing regions in inverse relation to the distance from the lesion epicenter. The degra-dation of 68-kD NFP in SCI indicates the involvement of increased proteolytic


activity of calpain. The treatment of SCI rats with calpeptin (CP) and methylpred-nisolone (MP) prevents degradation of 68-kD NFP. In untreated rats, neuronal cellswith degraded 68-kD NFP and other cytoskeletal proteins are going to be structurallyunstable and, therefore, prone to activate cellular machinery leading to apoptosis.

PREVENTION OF APOPTOSIS IN SCI

As calpain is involved in the patholphysiology of SCI, an intense research efforthas been focused on examination of the effect of calpain inhibitors as therapeutic

FIGURE 1. Extent of 68-kD NFP degradation in five different areas in SCI rat. Injury(40 g-cm) was induced on T12 by weight-drop. Calpain inhibitor CP (250 µg/kg) and anti-inflammatory agent MP (165 mg/kg) in 1.5% dimethyl sulfoxide (DMSO) as vehicle wereadministered intravenously within 30 min after injury. Following treatment, rats were sac-rificed at 48 hr. A 5-cm section of spinal cord with lesion in the center was removed anddivided into five 1-cm segments. Western analysis with proteins from SCI segments showed68-kD NFP degradation in untreated (vehicle) SCI rats (n = 7), and prevention of 68-kD NFPdegradation in drug-treated SCI rats (n = 9). Band intensities of 68-kD NFP were quantitateddensitometrically using PDI Quantity One software. Percent loss of 68-kD NFP in SCI ratswith or without treatment was calculated with respect to sham (uninjured) rats (n = 4). Dataare presented as mean + SEM of separate expreiments.


agents in SCI. Calpastatin (110 kD), the natural inhibitor of calpain, is too large tobe cell permeable. With an increased calpain:calpastatin ratio, calpastatin is degrad-ed as a suicide substrate of calpain and, therefore, becomes ineffective. A cysteineresidue (Cys108) at the catalytic site places calpains in the family of cysteine pro-teases. The thiol group (-SH) of Cys108 directly participates in covalent catalyticcleavage of peptide bond of the substrate. The inhibitors of calpain act by covalentinteraction between the -SH of the active site Cys108 and an electrophilic center ofthe inhibitor. Calpeptin (CP), a dipeptide aldehyde, has been reported to inhibit pro-teolytic activity of calpain.45 As CP is a small compound lacking charged groups, itis capable of penetrating the cell membrane by passive diffusion. Free radicals aregenerated by activation of inflammatory cells in SCI and, therefore, an antiinflam-

FIGURE 2. Extent of internucleosomal DNA fragmentation in five different areas inrat SCI. Induction of SCI, treatment of SCI rats, and collection of SCI segments are the sameas described in FIGURE 1. Genomic DNA samples extracted from SCI segments were re-solved on a 1.6% agarose gel by electrophoresis. M, marker (1 kb ladder); vehicle, DNAfragmentation occurred maximally in lesion (lane 3), and minimally in rostral areas (lanes1 and 2) and caudal areas (lanes 4 and 5) of the spinal cord from untreated SCI rat; treat-ment, DNA fragmentation was prevented in all five segments of the spinal cord from drug-treated SCI rat. Results are representative of three separate experiments. There was no in-ternucleosomal DNA fragmentation in these segments of the spinal cord from sham rat (datanot shown).


matory agent may ameliorate neural cell death in SCI. Glucocorticoids includingmethylprednisolone (MP) are antiinflammatory, and have been used in the treatmentof SCI and inflammatory diseases such as multiple sclerosis. The exact mechanismof antiinflammatory action of glucocorticoids is not fully understood, but it is partlydue to inhibition of PLA2,46 which catalyzes the release of arachidonic acid frommembrane phospholipids with subsequent production of free radicals. The inductionof inflammatory processes in SCI warranted the therapeutic use of a potent antiin-flammatory agent such as MP, and its administration was neuroprotective in experi-mental and acute SCI.47,48 Pharmacological studies suggest that MP is aneuroprotective agent.49 Our recent in vitro study indicated a new mechanism of MPaction with inhibition of calpain activity.50 To a large extent, calpain and inflamma-tory mediators cause cell death and tissue destruction in SCI, and hence a therapeuticregimen containing a calpain inhibitor and an antiinflammatory agent may preventapoptotic death of neural cells in SCI. Since many secondary destructive pathwaysare involved in tissue destruction in SCI, treatment with one therapeutic agent maynot be highly effective. A combination therapy with calpain inhibitor CP and freeradical inhibitor MP may have more synergistic neuroprotective effect than therapywith either one alone. We have investigated the efficacy of this combination therapyin SCI rats, and found that coadministration of CP (to inhibit calpain activity) andMP (to inhibit inflammatory process) in rat immediately after SCI reduced the extentof internucleosomal DNA fragmentation (apoptosis) in the lesion site and adjacentareas (FIG. 2). These results suggest that prevention of calpain-mediated protein deg-radation and inhibition of inflamatory process confer neuroprotection by preventionof apoptosis in rat SCI. The coadministration of CP and MP was more effective forinhibition of apoptosis than administration of CP or MP alone (data not presented).

CONCLUDING REMARKS

SCI initiates a complex neuropathological process stemming from a rapid in-crease in intracellular free Ca2+ and production of free radicals. These cause activa-tion of Ca2+-dependent proteases including calpain, and the progressive injuryultimately results in the loss of axon-myelin structural unit, degradation of cytoskel-etal proteins, and massive apoptotic and also necrotic death of neural cells. Untreat-ed SCI severely impairs motor function because of myelin loss, axonal degeneration,and neuronal cell death. The degradation of 68-kD NFP indicated the increasedcalpain activity in SCI lesion and adjacent areas, while the free radicals generatedby inflammatory processes contribute to upregulation of intracellular free Ca2+ andseveral biochemical pathways to mediate cell death in the CNS. In an attempt to pre-vent apoptosis caused by calpain and free radicals in rat SCI, administration of CPand MP within 30 min of injury is found promising, and we think that inhibition ofapoptosis by combination of CP and MP may help restore motor function in SCI.

ACKNOWLEDGMENTS

This investigation was supported in part by grants from the NIH-NINDS, the Na-tional MS Society, and the American Health Assistance Foundation.


REFERENCES

1. ZHIVOTOVSKY, B., D.H. BURGESS, D.M. VANAGS & S. ORRENIUS. 1987. Involvement ofcellular machinery in apoptosis. Biochem. Biophys. Res. Commun. 230: 481–488.

2. LINNEK, M.D., R.H. ZOBRIST & M.D. HATFIELD. 1993. Evidence supporting a role forprogrammed cell death in focal cerebral ischemia in rats. Stroke 24: 2002–2028.

3. BERKELAAR, M., D.B. CLARKE, Y.C. WANG, G.M. BRAY & A.J. AGUAYO. 1994. Axot-omy results in delayed death and apoptosis of retinal ganglion cells in adult rats. J.Neurosci. 14: 4368–4374.

4. CARAFOLI, E. & M. MOLINARI. 1998. Calpain: a protease in search of a function? Bio-chem. Biophys. Res. Commun. 247: 193–203.

5. TRUMP, B.F. & I.K. BEREZESKY. 1995. Calcium-mediated cell injury and cell death.FASEB J. 9: 219–228.

6. NICOTERA, P. & S. ORRENIUS. 1998. The role of calcium in apoptosis. Cell Calcium 23:173–180.

7. HAPPEL, R.D., K.P. SMITH, N.L. BANIK, J.M. POWERS, E.L. HOGAN & J.D. BALENTINE.1981. Ca2+-accumulation in experimental spinal cord trauma. Brain Res. 211: 476–479.

8. SAIDO, T.C., M. YOKOTA, S. NAGAO, I. YAMAURA, E. TANI, T. TSUCHIYA, K. SUZUKI &S. KAWASHIMA. 1993. Spatial resolution of fodrin proteolysis in postischemic brain.J. Biol. Chem. 268: 25239–25243.

9. MARTIN, S.J., G.A. O’BRIEN, W.K. NISHIOKA, A.J. MCGAHON, A. MAHBOUBI, T.C.SAIDO & D.R. GREEN. 1995. Proteolysis of fodrin (non-erythroid spectrin) duringapoptosis. J. Biol. Chem. 270: 6425–6428.

10. JORDAN, J., M.F. GALINDO & R.J. MILLER. 1997. Role of calpain- and interleukin-1 βconverting enzyme-like proteases in the β-amyloid-induced death of rat hippocam-pal neurons in culture. J. Neurochem. 68: 1612–1621.

11. RAY, S.K., G.G. WILFORD, C.V. CROSBY, E.L. HOGAN & N.L. BANIK. 1999. Diversestimuli induce calpain overexpression and apoptosis in C6 glioma cells. Brain Res.829: 18–27.

12. TRAYSTMAN, R.J., J.R. KIRSCH & R.C. KOEHLER. 1991. Oxygen radical mechanisms ofbrain injury following ischemia and reperfusion. J. Appl. Physiol. 71: 1185–1195.

13. FOX, J.E., R.G. TAYLOR, M. TAFFAREL, J.K. BOYLES & D.E. GOLL. 1993. Evidencethat activation of platelet calpain is induced as a consequence of binding of adhe-sive ligand to the integrin, glycoprotein IIb-IIIa. J. Cell Biol. 120: 1501-1507.

14. INOMATA, M., M. HAYASHI, Y. OHNO-IWASHITA, S. TSUBUKI, T.C. SAIDO & S.KAWASHIMA. 1996. Involvement of calpain in integrin-mediated signal transduc-tion. Arch. Biochem. Biophys. 328: 129–134.

15. CROALL, D.E. & G.N. DEMARTINO. 1991. Calcium-activated neutral protease(calpain) system: structure, function, and regulation. Physiol. Rev. 71: 813–847.

16. SUZUKI, K. 1987. Calcium activated neutral protease: domain structure and activityregulation. Trends Biochem. Sci. 12: 103–105.

17. SUZUKI, K., S. IMAJOH, Y. EMORI, H. SAWASAKI, Y. MINAMI & S. OHNO. 1987. Cal-cium-acivated neutral protease and its endogenous inhibitor. Activation at the cellmembrane and biological functions. FEBS Lett. 220: 271–277.

18. PONTREMOLI, S., F. SALAMINO, B. SPARATORE, R. DE TULLIO, R. PONTREMOLI & E. MEL-LONI. 1988. Characterization of the calpastatin defect in erythrocytes from patientswith essential hypertension. Biochem. Biophys. Res. Commun. 157: 867–874.

19. PONTREMOLI, S., E. MELLONI, P.L. VIOTI, M. MICHETTI, F. SALAMINO & B.L.HORECKER. 1991. Identification of two calpastatin forms in rat skeletal muscle andtheir susceptibility to digestion by homologous calpains. Arch. Biochem. Biophys.288: 646–652.

20. LI, Z., E.L. HOGAN & N.L. BANIK. 1995. Role of calpain in spinal cord injury:increased mcalpain immunoreactivity in spinal cord after compression injury in therat. Neurochem. Int. 27: 425–432.


21. GIULAN, D. & L.B. LACHMAN. 1985. Interleukin-1 stimulation of astroglial prolifera-tion after brain injury. Science 228: 497–499.

22. OTT, L., C.J. MCCLAIN, M. GILLESPIE & B. YOUNG. 1994. Cytokines and metabolicdysfunction after severe head injury. J. Neurotrauma 11: 447–472.

23. PERRY, V.H. 1994. Macrophage and microglia responses in CNS injury. In Macroph-ages and the Nervous System. V.H. Perry, Ed.: 62–86. R.G. Landes Company.Austin, TX.

24. LIPTON, S.A. & P. NICOTERA. 1998. Calcium, free radicals and excitotoxins in neu-ronal apoptosis. Cell Calcium 23:165–171.

25. HOGAN, E.L., W. MCIVER, A. KRALL & N.L. BANIK. 1985. Subcellular distribution ofcalcium in spinal cord trauma. Trans. Am. Soc. Neurochem. 16: 132.

26. GALLIN, J.I., I.M. GOLDSTEIN & R. SNYDERMAN. 1988. Inflammation: Basic Princi-ples and Clinical Correlates. Raven Press. New York.

27. HSU, C.Y., P.V. HALUSHKA, E.L. HOGAN, N.L. BANIK, W.A. LEE & P.L. PEROT, JR.1985. Alteration of thromboxane and prostacycline levels in experimental spinalcord injury. Neurology 35: 1003–1009.

28. BALENTINE, J.D. 1978. Pathology of experimental spinal cord trauma. Lab. Invest.39: 254–266.

29. MEANS, E.D. & D.K. ANDERSON. 1983. Neuronophagia by leukocytes in experimen-tal spinal cord injury. J. Neuropathol. Exp. Neurol. 42: 707–719.

30. XU, J.A., C.Y. HSU, T.H. LIU, E.L. HOGAN, P.L. PEROT, JR. & H.H. TAI. 1990. Leu-kotriene B4 release and polymorphonuclear cell infiltration in spinal cord injury. J.Neurochem. 55: 907–912.

31. KONTOS, H.A., E.P. WEI, J.T. POVLISHOCK & C.W. CHRISTMAN. 1984. Oxygen radi-cals mediate the cerebral arteriolar dilation from arachidonate and bradykinin incats. Circ. Res. 55: 295–303.

32. ANDERSON, D.K. & E.D. HALL. 1993. Pathophysiology of spinal cord trauma. Ann.Emerg. Med. 22: 987–992.

33. ELLIS, R.E., J. YUAN & H.R. HORVITZ. 1991. Mechanisms and functions of celldeath. Annu. Rev. Cell Biol. 7: 663–698.

34. THOMPSON, C.B. 1995. Apoptosis in the pathogenesis and treatment of disease. Sci-ence 267: 1456–1462.

35. FADEN, A.I. & R.P. SIMON. 1988. A potential role for excitotoxins in the pathophysi-ology of spinal cord injury. Ann. Neurol. 23: 623–626.

36. PANTER, S.S., S.W. YUM & A.I. FADEN. 1990. Alteration in extracellular amino acidsafter traumatic spinal cord injury. Ann. Neurol. 27: 96–99.

37. GWAG, B.J., J.Y. KOH, J.A. DEMARO, H.S. YING, M. JACQUIN & D.W. CHOI. 1997.Slowly triggered excitotoxicity occurs by necrosis in cortical cultures. Neuro-science 77: 393–401.

38. CROWE, M.J., J.C. BRESNAHAN, S.L. SHUMAN, J.N. MASTERS & M.S. BEATTIE. 1997.Apoptosis and delayed degeneration after spinal cord injury in rats and monkeys.Nat. Med. 3: 73–76.

39. LIU, X.Z., X.M. XU, R. HU, C. DU, S.X. ZHANG, J.W. MCDONALD, H.X. DONG, Y.J.WU, G.S. FAN, M.F. JACQUIN, C.Y. HSU & D.W. CHOI. 1997. Neuronal and glialapoptosis after traumatic spinal cord injury. J. Neurosci. 17: 5395-5406.

40. RAY, S., D. DAVIS, D. SHIELDS, D. MATZELLE, G. WILFORD, E.L. HOGAN & N.L.BANIK. 1998. Increased calpain expression, cell death, and neuroprotection in ratspinal cord injury. J. Neurochem. 70(Suppl. 1): S63.

41. BANIK, N.L., D.L. MATZELLE, G. GANTT-WILFORD, A. OSBORNE & E.L. HOGAN.1997. Increased calpain content and progressive degradation of neurofilament pro-tein in spinal cord injury. Brain Res. 752: 301–306.

42. RAY, S.K., D.C. SHIELDS, T.C. SAIDO, D.C. MATZELLE, G.G. WILFORD, E.L. HOGAN


& N.L. BANIK. 1999. Calpain activity and translational expression increased in spi-nal cord injury. Brain Res. 816: 375–380.

43. BAKI, A., P. TOMPA, A. ALEXA, O. MOLNAR & P. FRIEDRICH. 1996. Autolysis paral-lels activation of µcalpain. Biochem. J. 318: 897–901.

44. SAIDO, T.C., H. SORIMACHI & K. SUZUKI. 1994. Calpain: new perspectives in molec-ular diversity and physiological-pathological involvement. FASEB J. 8: 814–822.

45. TSUJINAKA, T., Y. KAJIWARA, J. KAMBAYASHI, M. SAKON, N. HIGUCHI, T. TANAKA &T. MORI. 1988. Synthesis of a new cell penetrating calpain inhibitor (calpeptin).Biochem. Biophys. Res. Commun. 153: 1201–1208.

46. HIRATA, F., E. SCHIFFMANN, K. VENKATASUBRAMANIAN, D. SALOMON & J. AXELROD.1980. A phospholipase A2 inhibitory protein in rabbit neutrophils induced by glu-cocorticoids. Proc. Natl. Acad. Sci. USA 77: 2533–2536.

47. BRAUGHLER, J.M. & E.D. HALL. 1984. Effects of multidose methylprednisolonesodium succinate administration on injured cat spinal cord neurofilament degrada-tion and energy metabolism. J. Neurosurg. 61: 290–295.

48. BRACKEN, M.B. 1991. Treatment of acute spinal cord injury with methylpredniso-lone: results of a multicenter, randomized clinical trial. J. Neurotrauma 8(Suppl.1): S47–S52.

49. HALL, E.D. 1991. The neuroprotective pharmacology of methylprednisolone. J. Neu-rosurg. 76: 13–22.

50. BANIK, N.L., D. MATZELLE, E. TERRY & E.L. HOGAN. 1997. A new mechanism ofmethylprednisolone and other corticosteroids action demonstrated in vitro: inhibi-tion of a proteinase (calpain) prevents myelin and cytoskeletal protein degradation.Brain Res. 748: 205–210.

calpeptin and methylprednisolone inhibit apoptosis in rat spinal cord injury

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