Immunohistochemical Study of Calpain-Mediated Breakdown Products to α-Spectrin Following Controlled...

JOURNAL OF NEUROTRAUMAVolume 14, Number 6, 1997Mary Ann Liebert, Inc.

Immunohistochemical Study of Calpain-Mediated BreakdownProducts to a-Spectrin Following Controlled Cortical Impact

Injury in the Rat

JENNIFER K. NEWCOMB, ANDREAS KAMPFL,1 RAND M. POSMANTUR,2XIURONG ZHAO, BRIAN R. PIKE, SHI-JIE LIU,

GUY L. CLIFTON, and RONALD L. HAYES

ABSTRACT

This study examined the effect of unilateral controlled cortical impact on the appearance of calpain-mediated a-spectrin breakdown products (BDPs) in the rat cortex and hippocampus at various timesfollowing injury. Coronal sections were taken from animals at 15 min, 1 h, 3 h, 6 h, and 24 h afterinjury and immunolabeled with an antibody that recognizes calpain-mediated BDPs to a-spectrin(Roberts-Lewis et al., 1994). Sections from a separate group of rats were also taken at the sametimes and stained with hematoxylin and eosin. Analyses of early time points (15 min, 1 h, 3 h, and6 h following injury) revealed a-spectrin BDPs in structurally intact neuronal soma and dendritesin cortex ipsilateral to site of injury that was not present in tissue from sham-injured control rats.By 24 h after injury labeling was not restricted to clearly defined neuronal structures in ipsilateralcortex, although there was an increased extent of diffuse labeling. BDPs to a-spectrin in axons werenot detected until 24 h after injury, in contrast to the more rapid accumulation of BDPs observedin neuronal soma and dendrites. The presence of BDPs to a-spectrin in the cortex at the site of im-pact, and in the rostral and contralateral cortex, coincided with morphopathology detected by hema-toxylin and eosin. a-Spectrin BDPs were also observed in the hippocampus ipsilateral to the injuryin the absence of overt cell death. This investigation provides further evidence that calpain is acti-vated after controlled cortical impact and could contribute to necrosis at the site of injury. The ap-pearance of calpain-mediated BDPs at sites distal to the contusion site and in the hippocampus alsosuggests that calpain activation may precede and/or occur in the absence of extensive mor-

phopathological changes.

Key words: calpain; a-spectrin; traumatic brain injury; cytoskeleton; immunohistochemistry

Vivian L. Smith Center for Neurologic Research, Department of Neurosurgery, University of Texas Houston Health ScienceCenter, Houston, TX 77030. 'Present address: University of Innsbruck, Department of Neurology, Intensive Care Unit, Anich-strasse 35, A-6020 Innsbruck, Austria, Europe. 2Present address: Parke-Davis Pharmaceuticals, Immunology/Neuroscience Ther-apeutics, 2800 Plymouth Road, Ann Arbor, MI 48105.

Abbreviations: TBI, traumatic brain injury; NF, neurofilament; MAP2, microtubule associated protein 2; BDPs, breakdownproducts; H&E, hematoxylin and eosin.

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INTRODUCTION

Loss of CYTOSKELETAL proteins, including neurofil-ament (NF) proteins, microtubule-associated protein

2 (MAP2), tau, and spectrin (fodrin), is an important char-acteristic in a variety of acute central nervous system(CNS) injuries including ischemia (Bartus et al, 1995;Kaku et al., 1993; Matesic and Lin, 1994; Nakamura etal., 1992; Roberts-Lewis et al., 1994), spinal cord injury(Banik et al., 1982), and traumatic brain injury (TBI) invivo (Hicks et al., 1995; Postmantur et al,. 1994, 1996b;Saatman et al., 1996a; Taft et al., 1992,1993) and in vitro(Kampfl et al., 1995, 1996b; Whitson et al., 1995a,b).

Several lines of evidence suggest that the pathologicalactivation of the calcium-dependent intracellular neutralproteases, calpains (Goll et al., 1990; Guroff, 1964; Mell-gren, 1980) may play a major role in cytoskeletal de-generation following acute brain injury, including TBI(for review, see Kampfl et al., 1997). Alterations in Ca2+homeostasis occur in TBI (Fineman et al., 1993; Nadleret al., 1995; Nilsson et al., 1993; Shapira et al., 1989) andischemia (Choi, 1988; Siesjoe and Bengtsson, 1989) asa result of excessive exposure to excitatory neurotrans-mitters (Faden et al,. 1989; for reviews, see Hayes et al.,1992; Mclntosh et al., 1996) and brief potassium depo-larization (Katayama et al., 1990). The two known isoen-zymes of calpain in the CNS, m-calpain (calpain 2) andiu,-calpain (calpain 1), are activated by mM and pM con-centrations of calcium, respectively (Goll et al. 1990).Autolysis of the 80-kD subunit to a 76-kD subunit, con-sidered to be an important event in the intramolecular ac-

tivation of /¿-calpain (Melloni and Pontremoli, 1989;Saido et al., 1994; Saito et al., 1993; Suzuki et al., 1981;Zimmerman and Schlaepfer, 1991), has been reportedfollowing TBI (Kampfl et al., 1996a) and ischemia (Neu-mar et al., 1996).

Substrates for calpain proteolysis include NF proteins(Banik et al., 1987; Schlaepfer and Zimmerman, 1985;Nixon et al., 1990), MAP2 (Johnson et al., 1991), andspectrin (Roberts-Lewis et al., 1994; Siman et al., 1984).Recent investigations have shown calpain-mediatedbreakdown products (BDPs) to neurofilament-light (NF-L) (Posmantur et al., 1994, 1997) and a-spectrin follow-ing TBI (Kampfl et al., 1996a; Posmantur et al., 1997;Saatman et al., 1996a) and ischemia (Roberts-Lewis etal., 1994; Saido et al., 1993). In addition, protease in-hibitors that block calpains have been shown to amelio-rate behavioral deficits in vivo (Markgraf, personal com-

munication; Saatman et al., 1996b) and attenuatecytoskeletal protein loss in in vitro (Brorson et al, 1995;George et al., 1995; Kampfl et al., 1995, 1996b) and invivo (Posmantur et al,. 1997) models of TBI. These pro-tease inhibitors have also been shown to diminish cellu-

lar damage following ischemia (Bartus et al., 1994; Honget al., 1994; Inuzuka et al., 1990; Lee et al., 1991; Ramiand Krieglstein, 1993). Because none of these proteaseinhibitors are solely selective for calpains (Wang andYuen, 1994), and some may also inhibit other cysteineproteases (Saatman et al., 1996b), these data provide onlyindirect evidence for calpain activation following TBIand ischemia.

Spectrin is a substrate for calpain (Siman et al., 1984)and is frequently used as a biochemical marker for cal-pain proteolysis (Blomgren et al., 1995; Nixon, 1986;Roberts-Lewis et al., 1994; Saido et al., 1993; Siman etal., 1989). This protein is an integral component of thecytoskeleton, underlying the plasma membrane and form-ing a structural lattice. Spectrin is a heterodimer com-

posed of two large polypeptide chains, a (~240,000 kD)and ß (~235,000 kD), which self-associate head-to-headto form tetramers (Dhermy, 1991). Previous Westernblotting studies in our laboratory employing an antibodythat recognizes calpain-mediated BDPs to a-spectrin(Roberts-Lewis et al., 1994) have shown accumulation ofthese BDPs in the cortex ipsilateral to the site of a cor-tical impact injury in the rat (Kampfl et al., 1996a; Pos-mantur et al., 1997). A recent study by Saatman et al.(1996a), employing a lateral fluid-percussion (FP) injurymodel, reported accumulation of calpain-mediated BDPsto a-spectrin within 90 min of injury using immunohis-tochemistry and within 24 h using Western blots. How-ever, regional and cellular distributions of calpain-medi-ated BDPs in the lateral controlled cortical impact model(CCI; Dixon et al., 1991) of TBI are unknown. It is im-portant to characterize changes in calpain-mediated BDPsfollowing cortical impact since this model employs dif-ferent biochemical inputs than FP injury, and unlike fluid-percussion injury, can be associated with ischémie re-duction in cerebral blood flow (CBF) (Bryan et al., 1995;Yamakami and Mclntosh, 1991; also see Dixon andHayes, 1996, for review). Thus, the present study em-

ployed immunohistochemistry and sought to determine:(1) whether calpain-mediated BDPs to a-spectrin were

primarily restricted to the area of contusion seen at thesite of cortical impact injury or extended to cortical re-

gions outside of the focal contusion associated with in-jury; or (2) whether calpain-mediated BDPs were de-tected in the dorsal hippocampus, a site not associatedwith significant morphopathology at the injury level em-

ployed in this study. The study focused on time pointsbetween 15 min and 24 h, as previous studies in our lab-oratory suggested that calpain was activated within thistime period (Kampfl et al., 1996a; Postmantur et al.,1994, 1997).

The results of the present immunohistochemical in-vestigation indicate that calpain proteolysis of a-spectrin

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CALPAIN-MEDIATED BREAKDOWN PRODUCTS TO a-SPECTRIN FOLLOWING TBI

occurs within minutes of controlled cortical impact TBI(<15 min) at the site of impact and precedes the com-

plete evolution of neuronal degeneration and necrosis thatis characterized by tissue loss, fragmentation, and cavi-tation. Calpain proteolysis of a-spectrin can occur in re-

gions distal to the site of injury that are not associatedwith overt, focal necrosis and contusion. In addition, our

results show a delayed appearance of BDPs to a-spectrinin axons, in contrast to the more rapid accumulation ofBDPs observed in neuronal soma and dendrites.

MATERIALS AND METHODS

SubjectsAdult male Sprague-Dawley rats weighing between

250 and 275 g were used. Animals were housed in cageswith food and water continually available. Animals were

divided into groups of naive, sham-injured, and injuredfor the following time points: 15 min, 1 h, 3 h, 6 h, and24 h (n = 4-5 per time point, per stain). Separate groupsof animals were used for a-spectrin breakdown productimmunohistochemistry or hematoxylin and eosin (H&E)staining. After injury, animals were housed in a separatecage from uninjured rats. All animal studies carefullyconformed to the guidelines outlined in the Guide for theCare and Use of Laboratory Animals from the U.S. De-partment of Health and Human Services and were ap-proved by the University of Texas—Houston Health Sci-ence Center Animal Welfare Committee.

- FIG. 1. Sections (—3.4 mm Bregma) stained with hematoxylinand eosin to assess morphopathology at the site of impact. Sham-injured brains (A, 100X; B, 320X) revealed no detectablehistopathological alterations. Injured rat cortex ipsilateral to siteof injury at 15 min (C, 100X; D, 320X), 1 h (E, 100X; F, 320X),3 h (G, ÎOOX; H, 320X), 6 h (I, lOOX; J, 320X), and 24 h (K,100X; L, 320 X) after TBI revealed evolutionary changes in themorphology of the contusion site. Photomicrographs of ipsilat-eral cortex at 15 min (C, D) and 1 h (E, F) after TBI showedfocal subpial hemorrhage (closed arrows) and the appearance ofeosinophilic neurons (curved arrows). Low magnification mi-crographs (100X) at 3 h (G) and 6 h (I) after TBI revealed in-tracortical hemorrhage (closed arrows) and an area of necrotictissue extending transversely from the site of injury. By 24 h(K) following TBI, this area of necrotic tissue had expanded.Higher magnification micrographs (320X) at 3 h (H), 6 h (J),and 24 h (L) after TBI demonstrated a progressive increase inpyknotic, shrunken triangular neurons (curved arrows) that was

associated with an increasing amount of cytoplasmic retraction(thin arrows) and a more extensive vacuolar appearance to theneuropil. Bars = 130 pm (A); 45 pm (B).

Production of Cortical Impact Brain InjuryThe controlled cortical impact device (Dixon et al.,

1991) consisted of a small (1.975 cm) bore, double-act-ing, stroke-constrained, pneumatic cylinder with a 5.0-cmstroke. The cylinder was rigidly mounted in a vertical po-sition on a crossbar, which could be precisely adjusted inthe vertical axis. The lower rod end had an impactor tipattached (i.e., that part of the shaft that comes into con-

tact with the exposed dura mater). The upper rod end was

attached to the transducer core of a linear velocity dif-ferential transducer (LVDT) (Shaevitz Model 500 HR),which produces an analog signal that was recorded by a

PC-based data acquisition system (R.C. Electronics) for

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analysis of time/displacement parameters of the impactor.The velocity of the impactor shaft was controlled by gaspressure.

All animals were initially anesthetized with 4% isoflu-rane with a 2:1 N2O/O2 mixture in a vented anesthesiachamber. Following endotracheal intubation, rats were

mechanically ventilated with a 2% isoflurane mixture.The rats were then mounted in the stereotaxic frame in a

prone position secured by ear and incisor bars. The headwas held in a horizontal plane with respect to the in-teraural line. Two 7-mm craniotomies were made be-tween lambda and bregma, centered 5 mm laterally on

either side of the central suture. After bilateral cran-

iotomy, the injury was induced.The animals received a cortical impact through the

craniotomy at a velocity of 6 m/sec. The injury devicewas set to produce a tissue deformation of 2 mm. Thedura was kept intact. Sham rats underwent identical sur-

gical procedures, but were not injured. Core body tem-

perature was monitored continuously by a rectal ther-mistor probe and maintained at 37-38°C. After injury,the scalp was sutured closed, anesthesia was discontin-ued, and the animal was extubated and assessed for re-

covery of reflexes (Dixon et al., 1991). Animals whoserighting response exceeded 5 min from time of impactwere omitted from the study. Following recovery fromanesthesia, animals were monitored visually and returnedto cages where they received normal access to food andwater.

a-Spectrin ImmunohistochemistryPrior to perfusion all animals were given a lethal dose

of pentobarbital (100 mg/kg i.p.). Transcardial perfusionwas administered through the left ventricle of the heartwith 120 ml of 0.9% saline and 200 ml of 4%paraformaldehyde in PBS (136-mM NaCl, 81-mM KC1,1.6-mM Na2HP04, and 14-mM KH2P04, pH 7.4) at theabove time points following injury. After injury, brainswere removed and postfixed for 3-^1 h in 4%paraformaldehyde. Brains were then soaked in a 30% su-

crose/PBS solution at 4°C overnight for adequate cry-oprotection. Each brain was grossly sectioned, frozen,and mounted in a cryostat (Leica CM 1800). Coronal sec-

tions of —40 fim thickness were cut at—

18°C, immedi-

M-FIG. 2. Sections (—3.4 mm Bregma) stained with hema-toxylin and eosin to assess morphopathology contralateral to thesite of impact. Sham-injured brains (A, 100X; B, 320X) re-

vealed no detectable histopathological alterations in either cor-

tex. Injured rat cortex contralateral to the site of injury at 15min (C, 100X; D, 320X), 1 h (E, 100X; F, 320X), 3 h (G,ÎOOX; H, 320X), 6 h (I, lOOX; J, 320X),. and 24 h (K, 100X;L, 320 X) revealed progressive changes in morphopathology.Photomicrographs of contralateral cortex at 15 min (C, D) and1 h (E, F) following TBI showed a small number of eosinophilicneurons (curved arrows). Photomicrographs of contralateral cor-

tex at 3 h (G, H), 6 h (I, J), and 24 h (K, L) revealed an in-crease in pyknotic, shrunken triangular neurons (curved arrows)that was associated with cytoplasmic retraction (straight arrows)and a more extensive vacuolar appearance to the neuropil. Theevolution of morphopathological changes was similar but lesspronounced in the contralateral cortex, compared to thosechanges seen in the ipsilateral cortex at the site of impact.Bars = 130 /xm (A); 45 pm (B).

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ately placed in wells containing PBS, and stored at 4°C.Sections located at approximately +0.02 mm (rostral tothe site of impact) and

—

3.4-mm Bregma (site of impact)were used to assess the presence of calpain-mediated a-

spectrin BDPs. The entire immunolabeling procedurewas performed in 6-well culture plates (Corning). Sec-tions were first blocked in 5% normal goat serum (Vec-tor) in TBS (20-mM Tris-HCl and 150-mM NaCl, pH7.5) with 0.1% Triton X-100. Sections were incubated inprimary antibody overnight at 4°C with continuous agi-tation (1:15,000) (Ab38, a generous gift of Dr. RobertSiman, Cephalon Inc., West Chester, PA, USA). This an-

tibody recognizes the cleavage site CQQQEVY and rec-

ognizes calpain-mediated BDPs to a-spectrin (Roberts-Lewis et al., 1994). Sections were then transferred to a

biotinylated goat anti-rabbit IgG (1:200) and incubatedfor 60 min (Vectastain rabbit Elite ABC kit), followedby incubation in a solution of avidin-biotin-peroxidasecomplex (1:100) for 60 min (Vectastain Elite ABC kit).The sections were rinsed, mounted onto slides, and vi-sualized with diaminobenzidine (DAB). All slides wereincubated in DAB for approximately 2 min. Sectionswere dehydrated through a series of graded ethanol so-lutions and coverslipped with Permount. Control sectionsincubated without primary antibody (Ab38) were not im-munoreactive.

Hematoxylin and Eosin StainingHematoxylin and eosin (H&E) stained sections were

used to assess morphological changes following CCI in-jury. Neurons undergoing morphopathological changesappeared dark, shrunken, and eosinophilic. The neurophil

- FIG. 3. Sections (+0.2 mm Bregma) stained with hema-toxylin and eosin to assess morphopathology in cortex rostralto the site of impact. Sham-injured brains (A, 100X; B, 320X)revealed no detectable histopathological alterations. Injured ratcortex rostral to the site of injury at 15min(C, 100X;D, 320X),1 h (E, 100X; F, 320X), 3 h (G, ÎOOX; H, 320X), 6 h (I, ÎOOX;J, 320 X), and 24 h (K, 100X; L, 320 X) after TBI revealedslight alterations in morphology. Photomicrographs of rostralcortex at 15 min (C, D), 1 h (E, F), and 3 h (G, H) followingTBI revealed a number of eosinophilic neurons (curved arrows).Photomicrographs taken 6 h (I, J) and 24 h (K, L) followingTBI revealed an increased number of pyknotic, shrunken tri-angular neurons (curved arrows) that was associated with cy-toplasmic retraction (straight arrows) and an extensive vacuo-lar appearance to the neuropil. The evolution ofmorphopathological changes was similar, but less pronouncedin the rostral cortex, compared to those changes seen in the ip-silateral cortex at the site of impact. Bars = 130 pm (A); 45pm (B).

was also characterized by a vacuolar appearance. Thesechanges occurred prior to focal, overt necrosis, charac-terized by tissue loss, fragmentation, and cavitation.

Animals were given a lethal dose of pentobarbital (100mg/kg i.p.) prior to perfusion. Rats were transcardiallyperfused through the left ventricle (120 ml 0.9% salineand 200 ml 10% buffered formalin) at the selected timepoint after injury. Rat brains were removed and postfixedin 10% buffered formalin overnight. Brains were grosslysectioned coronally at 2-3-mm intervals and processedthrough graded alcohols and xylenes. Brains were thenembedded in paraffin and cut into 4-5-pm sections us-

ing a microtome. Paraffin sections from brain regions

—,

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-*a e

+0.2 mm (rostral to site of impact) and —3.4 mm Bregma(site of impact) were mounted on slides and stained withH&E to assess neuronal morphopathology. Paraffin sec-

tions were used to obtain thin sections and optimal H&Estaining.

RESULTS

CCI Results in Evolutionary HistopathologicalChanges in Ipsilateral Cortex at the Site of Injury

Sham and naive brains (Fig. 1A, B) revealed no de-tectable histopathological alterations in either cortical

FIG. 4. Sections (-3.4 mm Bregma) immunolabeled withAb38, an antibody which recognizes calpain-mediated break-down products to a-spectrin. Sham-injured brains showed no

specific immunolabeling (A, 100X; B, 320X) in cortex. Injuredrat cortex ipsilateral to the site of injury at 15 min (C, 100X;D, 320X), 1 h (E, 100X; F, 320x), 3 h (G, ÎOOX; H, 320X),6 h (I, lOOX; J, 320X), and 24 h (K, 100X; L, 320X) after TBIrevealed prominent labeling of breakdown products. Low-mag-nification micrographs (C, E, G, I, and K) showed the area ofcontusion and diffuse, but specific immunolabeling, that ex-

tended into deeper cortical layers as time progressed. High mag-nification micrographs (D, F, H, J, and L) revealed structurallyintact neuronal cell bodies (curved arrows) at 15 min (D) andcontinuous dendrites (straight arrows) at 1 h (F) and 3 h (H)following TBI, labeled specifically for a-spectrin breakdownproducts. However, by 6 h post-TBI (J), some dendrites pos-sessed slightly discontinuous and fragmented labeling (straightarrows). At 24 h (L) following TBI, there were no clearly de-fined neuronal structures in the ipsilateral cortex, although therewas an increased extent of diffuse labeling not restricted to cellbodies. Bars = 130 pm (A); 45 pm (B).

hemisphere following perfusion and brain removal. At 15min following TBI (Fig. IC, D), tissue at the site of con-

tusion (—

3.4 mm Bregma) was characterized by focalsubpial hemorrhage and the appearance of eosinophilicneurons. These neurons were scattered throughout corti-cal layers I-V within an area extending approximately1-2 mm transversely from the site of injury. By 1 h post-TBI (Fig. IE, F), the number of eosinophilic neurons inthe cortex had increased. At 3 h (Fig. IG, H) and 6 h(Fig. II, J) post-TBI, H&E staining revealed intracorti-cal hemorrhage, an expanded area of damage in the cor-

tex, and additional pathological changes in neuronalstructure. Pyknotic, shrunken triangular neurons were ob-served and associated with an increasing amount of cy-toplasmic retraction and a more extensive vacuolar ap-pearance of the neuropil in cortical layers I-VI, whichwas also observed at 24 h post-TBI (Fig. IK, L). Fur-thermore, by 24 h post-TBI, the damaged cortical areahad extended from approximately 1-2 mm seen at 15 minpostinjury to 3-4 mm, and appeared overtly necrotic incortical layers I-VI. At all the time points examined, thisprimary injury zone revealed a gradual progression tonormal cortex on either side with decreasing numbers ofeosinophilic neurons.

CCI Results in Less Severe HistopathologicalChanges in Cortex Contralateral to the Siteof Injury

Sham and naive brains (Fig. 2A, B) revealed no detectablehistopathology in the cortex contralateral to the site of in-

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HFIG. 5. Section (—3.4 mm Bregma) immunolabeled with Ab38, an antibody that recognizes calpain-mediated breakdown prod-ucts to a-spectrin. Photomicrograph of a neuron 3 h following TBI in cortex ipsilateral to site of injury. Processes appeared in-tact, although the photomicrograph reveals some punctate labeling in the upper right corner. Bar = 70 pm.

jury (—3.4 mm Bregma). Fifteen minutes following TBI(Fig. 2C, D), the injured contralateral cortex revealed a smallnumber of eosinophilic neurons located primarily through-out cortical layers I—II, which extended into layer VI by 1h (Fig. 2E, F). At 3 h (Fig. 2G, H) and 6 h (Fig. 21, J) fol-lowing injury, H&E staining revealed additional pathologi-cal changes in neuronal structure, which were more evidentby 24 h post-TBI (Fig. 2K, L). Pyknotic, shrunken triangu-lar neurons were observed and associated with a largeamount of cytoplasmic retraction and a more extensive vac-

uolar appearance of the neuropil in cortical layers I-VI. Atall time points studied, the primary area of damage in thecontralateral cortex revealed a gradual progression to nor-

mal cortex on either side with decreasing numbers ofeosinophilic neurons. The evolution of morphopathologicalchanges was similar, but less pronounced compared to thosechanges seen in the ipsilateral cortex at the site of impact.

CCI Results in Less Severe HistopathologicalChanges in Ipsilateral Cortex Rostral to the Siteof Injury

Sham and naive brains (Fig. 3A, B) revealed no de-tectable histopathology in the ipsilateral rostral cortex(+0.2 mm Bregma). Fifteen minutes following TBI (Fig.3C, D), a small number of eosinophilic neurons was ob-

FIG. 6. Sections immunolabeled with Ab38, an antibody that recognizes calpain-mediated breakdown products to a-spectrin,15 min following TBI. Photomicrographs of cortex rostral (A, +0.2 mm Bregma) and contralateral (B, -3.4 mm Bregma) to thesite of injury revealed specific dendritic labeling for calpain-mediated breakdown to a-spectrin (arrows). Labeling was restrictedto superficial layers of cortex and occurred in the absence of focal necrosis.

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FIG. 7. Sections (-3.4 mm Bregma) immunolabeled withAb38, an antibody that recognizes calpain-mediated breakdownproducts to a-spectrin. Photomicrographs taken 3 h (A, 100X;B, 320X) after TBI revealed specific neuronal labeling in thedorsal hippocampus in the absence of focal necrosis. Neuronswere stained diffusely throughout the dorsal hippocampus, butconcentrated in the hilus of the dentate gyrus (A, B) and subicu-lum (not shown). Bars = 130 jam (A); 45 pm (B).

served in cortical layers I-II. By 1 h (Fig. 3E, F) and 3 h(Fig. 3G, H) post-TBI, the number of eosinophilic neu-rons had increased and extended into deeper cortical lay-ers (I-VI). At 6 h (Fig. 31, J) and 24 h (Fig. 3K, L) post-TBI, additional pathological changes were revealed.Pyknotic shrunken neurons were observed and associatedwith cytoplasmic retraction. Furthermore, there was avacuolar appearance of the neuropil. Overall, the amountof morphopathology observed was less severe in the cor-tical area rostral to the site injury (+0.2 mm Bregma)compared with the area of focal contusion and hemor-rhage (—3.4 mm Bregma). In addition, the damaged area

was limited to a smaller region compared to the tissue atthe site of impact.

CCI Results in Accumulation of BreakdownProducts to a-Spectrin in Ipsilateral Cortex atthe Site of Injury

The presence of calpain-mediated BDPs to a-spectrinfollowing TBI was examined using immunohistochem-istry. No localized immunoreactivity was present in tis-sue from sham-injured or naive control rats (Fig. 4A, B).BDPs to a-spectrin were observed at the site of injury(-3.4 mm Bregma) at all time points studied. The pri-mary site of immunoreactivity was in the ipsilateral cor-

tex, at the site of contusion and hemorrhage. At 15 minpost-TBI (Fig. 4C, D), BDPs to a-spectrin were seen pri-marily in neuronal cell bodies in the superficial corticallayers. Importantly, these prominent BDPs were observedin the absence of extensive morphopathological changes(Fig. IC, D: Discrepancies in tissue preservation in sec-

tions taken for H&E versus Ab38 immunohistochemistryare attributable to differences in tissue processing). Theipsilateral cortex at 1 h (Fig. 4E, F) post-TBI showedclear and evident staining in structurally intact neuronsin the cortex ipsilateral to site of injury. High magnifi-cation photomicrographs of the cortex 3 h after injury re-

vealed many intact processes, although the labeling of-ten appeared punctate (Fig. 4G, H; Fig. 5). By 6 hpost-TBI (Fig. 41, J), however, some dendrites possessedan increasingly fragmented appearance of immunolabel-ing compared to previous time points (Fig. 4E, F).

The characterization of immunoreactivity for calpain-mediated BDPs to a-spectrin evolved as time progressed.By 24 h post-TBI (Fig. 4K, L) there were no clearly de-fined neuronal structures in the ipsilateral cortex, althoughthere was an increased extent of diffuse labeling not re-

stricted to cell bodies. Immunolabeling at 24 h revealedpunctate-like staining in the ipsilateral cortex and severelyfragmented dendrites and neuronal soma, in contrast toearlier time points (15 min, 1 h, 3 h, and 6 h). Specificimmunoblotting was widespread and diffuse (Fig. 4E, G,I, and K). However, individual neurons and processes la-beled specifically with Ab38 were present (Fig. 4F, H, J,and L; see also Fig. 5). As time progressed (15 min-24 hpost-TBI), labeling extended into deeper cortical layers(I-IV; Fig. 4A, C, E, G, I, and K; see also Fig. 8C). Thecharacteristics of calpain-mediated BDPs to a-spectrinwas very consistent among animals at each time point.

Low Levels of Breakdown Products to tx-SpectrinExtend beyond Sites of Focal Contusion andNeuronal Necrosis

Calpain-mediated BDPs to a-spectrin were observedas early as 15 min following injury in the ipsilateral cor-

tex rostral to the injury site (+0.2 mm Bregma; Fig. 6A)

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:FIG. 8. Sections (—3.4 mm Bregma) immunolabeled with Ab38, an antibody that recognizes calpain-mediated breakdown prod-ucts to a-spectrin. Photomicrographs taken 3 h (A, 100X; B, 320 X) following TBI revealed diffuse, but specific, neuronal la-beling in the ipsilateral cortex. No axonal labeling was detected at this early time point. However, by 24 h (C, 100X; D, 320X)following TBI, the presence of punctate labeling in the corpus callosum (cc) was suggested. Bars = 130 pm (A); 45 pm (B).

and in the cortex contralateral to the site of impact (—

3.4mm Bregma; Fig. 6B). Neuronal labeling was diffuse andobserved in fine dendritic processes primarily in layers Iand II of the cortex. In addition, the magnitude and re-

gional extent of labeling did not differ dramatically be-tween time points (data not shown). Importantly, theBDPs seen at this time point occurred in the absence ofovert necrosis characterized by cavitation and tissue loss(Fig. 2C, D; Fig. 3C, D).

Subcortical structures, such as the dorsal hippocam-pus, also exhibited staining for BDPs to a-spectrin (Fig.7A, B). Neurons specifically labeled for BDPs were ob-served indiscriminately throughout the dorsal hippocam-pus in the absence of overt necrosis (data not shown). Ahigher concentration of labeled neurons, however, was

seen in the subiculum (data not shown) and in the hilusof the dentate gyrus (Fig. 7A, B). The magnitude of im-munolabeling did not differ dramatically between timepoints. Furthermore, the degree of immunoreactivity was

similar in both the ipsilateral and contralateral dorsal hip-pocampus. No immunoreactive neurons were detected insham or naive hippocampi.

CCI Results in Accumulation of BreakdownProducts to a-Spectrin in SubcorticalWhite Matter

BDPs to a-spectrin were observed in the corpus callo-sum at 24 h following TBI (Fig. 8C, D). Although specificneuronal labeling in cortical layer VI was evident at 3 hfollowing TBI, no axonal labeling was detected (Fig. 8A,B). In addition, no axonal labeling was detected 6 h fol-lowing injury (data not shown). Immunostaining in the cor-

pus callosum ipsilateral to the site of injury revealed axons

labeled in a fragmented and punctate fashion at 24 h post-TBI. No labeling was observed on the contralateral side.

DISCUSSION

This study provides the first characterization of thetemporal, regional, and cellular distributions of calpain-mediated BDPs to a-spectrin in the lateral controlled cor-tical impact model of TBI. Using immunohistochemistry,this study demonstrated that (1) calpain-mediated prote-

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olysis of a-spectrin occurred primarily at the site of con-tusion and, to a lesser extent, in areas distal to the site ofcontusion; (2) calpain-mediated BDPs to a-spectrin were

observed as early as 15 min at the site of cortical injuryprior to the onset of overt, focal necrosis characterizedby tissue loss, fragmentation, and cavitation, as well asin the cortex rostral and contralateral to the site of injury.By 24 h following injury, however, BDPs in the cortexwere associated with morphopathology detected by H&E;(3) calpain-mediated BDPs to a-spectrin were seen in thedorsal hippocampus at all time points examined in theabsence of overt cell death; (4) BDPs to a-spectrin were

observed in the axonal tracts of the corpus callosum ip-silateral to the site of contusion only at 24 h followinginjury. The delayed appearance of BDPs in this regioncontrasted with the rapid accumulation of BDPs in cellbodies and dendrites observed at early time points. Thesefindings suggest that calpain-mediated proteolysis of a-

spectrin precedes the full expression of morphopatho-logical changes, characterized by tissue loss, fragmenta-tion, and cavitation, that occur by 24 h in this model. Inaddition, these results demonstrate that dendrites and cellbodies may be more vulnerable than axons to calpain-mediated proteolysis at early time points following TBI.

Several lines of evidence suggest that activation of thecalcium-dependent proteases, caJpains, may play a promi-nent role in the pathology that results following acutetrauma such as TBI (for review, see Kampfl et al., 1997).Previous studies of excitotoxic mechanisms have reportedchanges in intracellular Ca2+ concentrations followingTBI (Fineman et al., 1993; Nadler et al., 1995; Nilsson etal., 1993; Shapira et al., 1989) and ischemia (Siesjoe andBengtsson, 1989). Cortical impact injury may also pro-duce focal ischemia in the cortex ipsilateral to the site ofinjury (Bryan et al., 1995), suggesting that reduced CBFmay also contribute to disturbances in intracellular Ca2+homeostasis and calpain activation following TBI. Fur-thermore, studies have reported that administration of cal-pain inhibitors can reduce loss of calpain substrates fol-lowing TBI (Posmantur et al., 1997) and ischemia (Bartuset al., 1994). These inhibitors, however, can also inhibitother cysteine proteases (Saatman et al., 1996b; Wang andYuen, 1994). Finally, studies employing TBI and ischemiamodels have observed BDPs to calpain substrates, suchas NF-L (Posmantur et al., 1994, 1997) and spectrin(Kampfl et al., 1996a; Posmantur et al., 1997; Saatman etal., 1996a; Roberts-Lewis et al., 1994; Saido et al., 1993)following injury. The present study investigated the re-

gional and cellular localization of calpain-mediated BDPsto a-spectrin following TBI across several time points us-

ing immunohistochemistry. The antibody employed inthis investigation recognized the calpain cleavage site on

a-spectrin and therefore labeled calpain-mediated BDPs

(Roberts-Lewis et al., 1994). Although this antibody hassome cross-reactivity with a-chymotrypsin cleaved a-

spectrin (Roberts-Lewis et al., 1994), a-chymotrypsin isnot an endogenous brain or blood protease.

Our results revealed evolutionary changes in calpain-mediated proteolysis of a-spectrin at the site of contu-sion. Calpain-mediated BDPs were detected in the cor-tex ipsilateral to the site of injury within 15 min afterinjury prior to the onset of overt, focal necrosis charac-terized by tissue loss, fragmentation, and cavitation. Pre-vious studies investigating a-spectrin proteolysis havealso observed BDPs at early time points in the absenceof overt, focal necrosis (Kampfl et al., 1996a; Saatmanet al., 1996a). However, by 24 h following injury, pres-ence of BDPs to a-spectrin coincided with overt necro-sis detected by H&E staining in the cortex ipsilateral tothe site of injury. These previous studies also reported a-

spectrin BDPs in conjunction with neuronal degenerationand morphopathology at the site of impact at later timepoints (Kampfl et al., 1996a; Saatman et al., 1996a). Stud-ies employing ischemia models have also reported celldeath at later time points following injury in associationwith calpain-mediated BDPs to a-spectrin (Blomgren etal., 1995; Roberts-Lewis et al., 1994; Yokotaetal., 1995).Ischemia results in preferential injury of cortical layersIII and IV (Brierly, 1976; Pulsinelli et al., 1982). Previ-ous studies in our laboratory have reported cytoskeletalloss, possibly related to calpain activation, in deeper cor-tical layers III-V (Posmantur et al., 1996). The presentstudy failed to detect increased immunoreactivity re-

stricted to specific cortical lamina. Reasons for this dis-crepancy are unclear. Calpain-mediated proteolysis of a-

spectrin also occurred in areas outside the area ofcontusion. Calpain-mediated BDPs were seen at all timepoints in the cortex contralateral and rostral to the site ofinjury. While evidence of proteolysis extended outsidethe regions of focal necrotic contusion, proteolysis in ros-

tral and contralateral brain regions occurred in associa-tion with more diffuse neuronal morphopathology evi-dent by 24 h after injury.

Calpain-mediated BDPs were also detected in the hip-pocampus. Although higher magnitudes of cortical im-pact injury have been reported to produce hippocampalcell loss (Cólicos et al., 1996; Cólicos and Dash, 1996;Goodman et al., 1994), the injury parameters employedin the present study did not result in overt hippocampalnecrosis at any of the time points examined. The ap-pearance of BDPs in an area not associated with cell deathsuggests that calpain activation may occur in the absenceof overt neuronal necrosis. Previous studies employingcentral FP have provided evidence of loss of MAP2, a

substrate for calpain, in the absence of neuronal cell death(Taft et al., 1992, 1993). It is still unclear as to why some

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cells undergoing proteolysis eventually die (corticalcells), while other cell populations undergoing proteoly-sis appear intact even at later time points (hippocampalcells). Hippocampal cells are preferentially more vulner-able in a variety of insults including hypoxia (Ng et al.,1989) and ischemia (Bonnekoh et al., 1990). Roberts-Lewis et al. (1994) proposed that only sustained calpainactivation leads to neuronal death following global is-chemia, suggesting that cortical cells undergo more sus-

tained calpain activation and proteolysis and eventuallydie. Other cell populations may undergo proteolysis forshorter periods of time, and therefore pathology may bereversible. Since the hippocampus is important for mem-

ory function, lethal or sublethal damage to hippocampalneurons could contribute to the memory deficits seen af-ter TBI in rats. Importantly, administration of a calpaininhibitor has been shown to protect against memorydeficits following lateral FP injury in rats (Saatman et al.,1996b).

An important finding in the present study was the de-layed proteolysis of a-spectrin in the corpus callosum 24h following injury, in contrast with the rapid accumula-tion of calpain-mediated BDPs in dendrites and soma.

Lateral FP injury results in accumulation of calpain-spe-cific BDPs to a-spectrin in intact axons of the corpus cal-losum within 90 min following injury, although by 24 hafter injury, axons appeared fragmented (Saatman et al.,1996a). A recent study reported preferential dendriticrather than axonal cytoskeletal damage 3 h following cor-

tical impact injury (Posmantur et al., 1996). In addition,studies looking at MAP2, a protein found primarily indendrites and soma (Bernhardt and Matus, 1984; Riedererand Matus, 1985), demonstrated MAP2 loss soon afterFP injury (Hicks et al., 1995; Taft et al., 1992). Previousstudies (Regehr and Tank, 1994; Yuste et al., 1994; Karstet al., 1993) have reported that dendrites and axons havedifferent electrophysiological properties which may ex-

plain the early cytoskeletal loss witnessed in dendritesfollowing TBI. Axons propagate neuronal transmissionby opening and closing Na+ and K+ channels, suggest-ing axons are less likely to experience large, immediatechanges in Ca+2 concentration. Dendrites, in contrast,propagate neuronal transmission by low-voltage Ca2+channels that may be more likely to open followingtrauma, therefore leading to the increased intracellularCa2+ concentration necessary for calpain activation.However, hypoxic injury to central axons, although lack-ing glutamate receptors, is still mediated by excess entryof Ca2+, perhaps occurring via the Na+-Ca2+ exchanger(Stys et al., 1993). There is also some evidence for pref-erential localization of m-calpain in axons, (Hamakuboet al., 1986; Li et al., 1995; Perlmutter et al., 1990), andthis isoform requires higher calcium concentrations for

activation than /¿-calpain (Goll et al., 1990). In addition,inhibitors of calpain (and other proteases) have been re-

ported to attenuate axonal damage following brain injuryin vivo (Posmantur et al., 1997) and in vitro (George etal., 1995; Kampfl et al., 1995,1996b). However, it is stillunclear whether proteolysis exclusively, or even primar-ily, contributes to axonal injury. Some investigators havesuggested that localized intra-axonal changes in cy-toskeletal alignment could lead to impairment in antero-grade transport resulting in axonal swelling and eventualdisconnection (Pettus et al., 1994; Povlishock et al., 1996;Yaghmai and Povlishock, 1992).

The present 5>tudy's findings are in general agreementwith a recent paper investigating calpain-mediated BDPsto a-spectrin following lateral fluid percussion injury(Saatman et al., 1996a). Both studies demonstrated thatthe primary areas which exhibited calpain-mediated pro-teolysis were the same areas which eventually revealedovert morphopathology. The two investigations also de-scribed structurally intact neuronal soma and dendriteslabeled for a-spectrin BDPs at early time points. How-ever, by 24 h following injury immunolabeling was morediffuse and revealed no discernible neuronal structures.

Finally, both studies observed a progression of specificimmunolabeling for calpain-mediated BDPs to a-spec-trin that began at superficial cortical layers and subse-quently extended into subcortical structures. However,the present study detected a somewhat different tempo-ral profile for a-spectrin proteolysis following corticalimpact injury than reported for lateral FP injury. Saat-man et al. (1996a) did not detect BDPs in gray matteruntil 90 min following injury, in contrast to detection ofBDPs within 15 min following cortical impact injury. Inaddition, Saatman et al. (1996a) reported a-spectrin pro-teolysis in the corpus callosum within 90 min followinginjury, while the present study did not detect such evi-dence until 24 h following cortical impact injury. Thereasons for these temporal discrepancies are not appar-ent and may be related to model differences. Secondaryischemia flow reductions could have contributed to pro-teolysis following cortical impact injury (Bryan et al.,1995), but not following lateral FP impact injury (Ya-makami and Mclntosh, 1991). The early onset of calpain-mediated proteolysis following cortical impact injury inthis study is in accordance with recent studies employ-ing the cortical impact model reporting increased calpainactivity within 15 min of injury measured either by cal-pain autolysis (Kampfl et al., 1996a) or casein zymogra-phy (Zhao et al., 1996).

In conclusion, the present investigation provides fur-ther evidence that calpain is activated following CCI in-jury and could contribute to necrosis at the site of injury.Calpain-mediated BDPs to a-spectrin were observed pri-

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marily at the site of contusion and to a lesser extent, inareas distal to the site of contusion. These BDPs were as-

sociated with overt morphopathology at later time points.In addition, calpain-mediated BDPs to a-spectrin wereseen in the dorsal hippocampus at all time points exam-ined in the absence of overt cell death. Finally, there was

a delayed appearance of BDPs to a-spectrin in the cor-

pus callosum ipsilateral to the site of contusion at 24 hfollowing injury. These findings suggest that calpain-me-diated proteolysis of a-spectrin precedes the full expres-sion of morphopathological changes, characterized by tis-sue loss, fragmentation, and cavitation, that occurs by 24h in this model. In addition, these results demonstrate thatdendrites and cell bodies may be more vulnerable to cal-pain-mediated proteolysis than axons at early time pointsfollowing injury.

ACKNOWLEDGMENTS

This work was supported by NIH grants5P01NS31998 and R01NS21458. Dr. A. Kampfl was

supported by a fellowship from the Austrian ScienceFoundation. The authors would like to thank Dr. R. Simanfor providing antibody 38, T. Gegeny and Dr. L. Jenkinsfor their critical review of the manuscript, and W. P. Flinnfor his technical assistance.

REFERENCES

BANIK, N.L., HAPPEL, R.D., SOSTEK, M.B., CHIU, F.C,and HOGAN, E.L. (1987). Ca2+-mediated degredation ofcentral nervous system (CNS) proteins: Topograhpic andspecies variation. Metab. Brain Dis. 2, 117-126.

BANIK, N.L., HOGAN, E.L., POWERS, J.M., and WHETS-TINE, L.J. (1982). Degradation of neurofilaments in spinalcord injury. Neurochem. Res. 7, 1465-1475.

BARTUS, R.T., DEAN, R.L., CAVANAUGH, K., EVELETH,D., CARRIERO, D.L., and LYNCH, G. (1995). Time relatedneuronal changes following middle cerebral artery occlusion:implications for therapeutic intervention and the role of cal-pain. J. Cereb. Blood How Metab. 15, 969-979.

BARTUS, R.T., HAYWARD, N.J., ELLIOTT, P.J., SAWYER,S.D., BAKER, K.L., DEAN, R.L., AKIYAMA, A.,STRAUB, J.A., HARBESON, S.L., LI, Z., and POWERS, J.(1994). Calpain inhibitor AK295 protects neurons from fo-cal brain ischemia: Effects of postocclusion intra-arterial ad-ministration. Stroke 25, 2265-2270.

BERNHARDT, R., and MATUS, A. (1984). Light and electronmicroscopic studies of the distribution of microtubule-asso-ciated protein 2 in rat brain: A difference between the den-dritic and axonal cytoskeletons. J. Comp. Neurol. 226,203-221.

BLOMGREN, K., MCRAE, A., BONA, E., SAIDO, T.C,KARLSSON, J-O., and HAGBERG, H. (1995). Degredationof fodrin and MAP2 after neonatal cerebral hypoxic-is-chemia. Brain Res. 684, 136-142.

BONNEKOH, P., BARBIER, A., OSCHLIES, U., and HOSS-MAN, K.-A. (1990). Selective vulnerability in the gerbil hip-pocampus: Morphological changes after 5-min ischemia andlong survival times. Acta Neuropathologica 80, 18-25.

BRIERLEY, J.B. (1976). Cerebral Hypoxia, in: Greenfield'sNeuropathology. W. Blackwood and J.A.N. Corsellis (eds),Edward Arnold Publishers: London, p. 56.

BRORSON, J.R., MARCUCILLI, C.J., and MILLER, R.J.(1995). Delayed antagonism of calpain reduces excitotoxic-ity in cultured neurons. Stroke 26, 1259-1267.

BRYAN, R.M., CHERIAN, L., and ROBERTSON, C. (1995).Regional cerebral blood flow after controlled cortical impactin rats. Anesth. Analg. 80, 687-695.

CHOI, D.W. (1988). Calcium mediated neurotoxicity: rela-tionship to specific channel type and role in ischémie dam-age. TINS 11, 465^169.

CÓLICOS, M.A., and DASH, P.K. (1996). Apoptotic mor-

phology of dentate gyms granule cells following experi-mental cortical impact injury in rats: Possible role in spatialmemory deficits. Brain Res. 739, 120-131.

CÓLICOS, M.A., DIXON, E.D., and DASH, P.K. (1996). De-layed, selective neuronal death following experimental cor-

tical impact injury in rats: possible role in memory deficits.Brain Res. 739, 111-119.

DHERMY, D. (1991). The spectrin super-family. Biol. of theCell 71, 249-254.

DIXON, CE., CLIFTON, G.L., LIGHTHALL, J.W., YAGH-MAI, A.A., and HAYES, R.L. (1991). A controlled corticalimpact model of traumatic brain injury in the rat. J. Neurosci.Methods 39, 253-262.

DIXON, CE., and HAYES, R.L. (1996). Fluid percussion andcortical impact models of traumatic brain injury, in Neuro-trauma. R.K. Narayan, J.E. Wilberger, and J.T. Povlishock(eds), McGraw-Hill: NY, pps. 1337-1346.

FADEN, A.I., DOGMATIC, P., PANTER, S.S., and VINK, R.(1989). The role of excitatory amino acids and NMDA re-

ceptors in traumatic brain injury. Science 244, 798-800.

FINEMAN, I., HOVDA, D., SMITH, M., YOSHINO, A., andBECKER, D. (1993). Concussive brain injury is associatedwith a prolonged accumulation of calcium. A 45Ca autoradi-ographic study. Brain Res. 624, 94—102.

GEORGE, E.B., GLASS, J.D., and GRIFFIN, J.W. (1995). Ax-otomy induced axonal degeneration is mediated by calciuminflux through ion-specific channels. J. Neurosci. 15,6445-6452.

GOLL, D.E., KLEESE, W.C, OKITANI, A., KUMAMOTO,T., CONG, J., and KAPPRELL, H-P. (1990). Historical

380


Background and Current Status of the Ca2+-Dependent Pro-teinase System, in: Intracellular Calcium-Dependent Prote-olysis. Mellgren, R.L., and Murachi, T. (eds), CRC Press:Boca Raton, FL, pps. 3-24.

GOODMAN, J.C., CHERIAN, L., BRYAN, JR., R.M., andROBERTSON, C.S. (1994). Lateral cortical impact injury inrats: Pathologic effects of varying cortical compression andimpact velocity. J. Neurotrauma 11, 587-597.

GUROFF, G. (1964). A neutral, calcium-activated proteinasefrom the soluble fraction of rat brain. J. Biol. Chem. 239,149-155.

HAMAKUBO, T., KANNAGI, R., MURACHI, T., and MA-TUS, A. (1986). Distribution of calpains I and II in rat brain.J. Neurosci. 6, 3103-3111.

HAYES, R.L., JENKINS, L.W., and LYETH, B.G. (1992).Neurotransmitter mediated mechanisms of traumatic braininjury. J. Neurotrauma 9, 173-187.

HICKS, R.R., SMITH, D.H., and McINTOSH, T.K. (1995).Temporal response and effects of excitatory amino acid an-

tagonism on microtubule-associated protein 2 immunoreac-tivity following experimental brain injury in rats. Brain Res.678, 151-160.

HONG, S.C., GOTO, Y., LANZINO, G, SOLEAU, S., KAS-SELL, N.F., and LEE, K.S. (1994). Neuroprotection with a

calpain inhibitor in a model of focal cerebral ischemia. Stroke25, 663-669.

INUZUKA, T, TAMURA, A., SATO, S., KIRINO, T,TOYOSHIMA, I., and MIYATAKE, T. (1990). Suppressiveeffect of E-64c on ischémie degradation of cerebral proteinsfollowing occlusion of the middle cerebral artery in rats.Brain Res. 526, 177-179.

JOHNSON, G.V.W., LITERSKY, J.M., and JOPE, R.S. (1991).Degradation of microtubule associated protein 2 and spectrinby calpain: a comparative study. J. Neurochem. 56,1630-1638.

KAKU, Y., YONEKAWA, Y., TSUKAHARA, T., OGATA,N, KTMURA, T., and TANIGUCHI, T. (1993). Alterationsof a 200kDa neurofilament in the rat hippocampus after fore-brain ischemia. J. Cereb. Blood Flow Metab. 13, 402^108.

KAMPFL, A., POSMANTUR, R., NIXON, R., GRYNSPAN,F., ZHAO, X., LIU, S.J., NEWCOMB, J.K., CLIFTON, G.L.,and HAYES, R.L. (1996a). /t-Calpain activation and calpain-mediated cytoskeletal proteolysis following traumatic braininjury. J. Neurochem. 67, 1575-1583.

KAMPFL, A., POSMANTUR, R.M., ZHAO, X.,SCHMUTZHARD, E., CLIFTON, G.L., and HAYES, R.L.(1997). Mechanisms of calpain proteolysis following trau-matic brain injury: Implications for pathology and therapy.A review and update. J. Neurotrauma, in press.

KAMPFL, A., WHITSON, S.J., ZHAO, X., POSMANTUR, R.,CLIFTON, G.L., and HAYES, R.L. (1995). Calpain in-hibitors reduce depolarization induced loss of tau protein in

primary septo-hippocampal cultures. Neurosci. Lett. 194,149-152.

KAMPFL, A., ZHAO, X., WHITSON, S.J., POSMANTUR, R,DIXON, CE., YANG, K, CLIFTON, G.L., and HAYES,R.L. (1996b). Calpain inhibitors protect against depolariza-tion induced neurofilament protein loss of septo-hippocam-pal neurons in culture. Eur. J. Neurosci. 8, 344—352.

KARST, H., JOELS, M., and WADMAN, W.J. (1993). Low-threshold calcium currents in dendrites of the adult rat hip-pocampus. Neurosci. Lett. 164, 154-158.

KATAYAMA, Y., BECKER, D.P., TAMURA, T., andHOVDA, D.A. (1990). Massive increases in extracellularpotassium and the indiscriminate release of glutamate fol-lowing concussive brain injury. J. Neurosurg. 73, 889-900.

LEE, K.S., FRANK, S., VANDERKLISH, P., ARAI, A., andLYNCH, G. (1991). Inhibition of proteolysis protects hip-pocampal neurons from ischemia. Proc. Nati. Acad. Sei. USA88, 7233-7237.

LI, Z., HOGAN, E.L., and BANIK, N.L. (1995). Role of cal-pain in spinal cord injury: Increased m-calpain immunore-activity in spinal cord after compression injury in the rat.Neurochem. Int. 27, 425^132.

MATESIC, D.F., and LIN, R.C.S. (1994). Microtubule associ-ated protein 2 as an early indicator of ischemia induced neu-

rodegeneration in the gerbil forebrain. J. Neurochem. 63,1012-1020.

McINTOSH, T.K, SMITH, D.H., and GARDE, E. (1996).Therapeutic approaches for the prevention of secondary braininjury. Eur. J. Anes. 13, 291-309.

MELLGREN, R.L. (1980). Canine cardiac calcium-dependentproteases: resolution of two forms with different require-ments for calcium. FEBS Lett. 109, 129-133.

MELLONI, E., and PONTREMOLI, S. (1989). The calpains.TINS 12, 438^144.

NADLER, V., BIEGON, A., BEIT-YANNAI, E., ADAM-CHIK, J., and SHOHAMI, E. (1995). 45Ca accumulation inrat brain after closed head injury; attenuation by the novelneuroprotective agent HU-211. Brain Res. 685, 1-11.

NAKAMURA, M., ARAKI, M., OGURO, K, and MA-SUZAWA, T. (1992). Differential distribution of 68kD and200kD neurofilament proteins in gerbil hippocampus andtheir early distributional changes following transient fore-brain ischemia. Exp. Brain Res. 89, 31-39.

NEUMAR, R.W., HAGLE, S.M., DEGARCIA, D.J.,KRAUSE, G.S., and WHITE, B.C. (1996). Brain /x-calpainautolysis during global cerebral ischemia. J. Neurochem. 66,421-424.

NG, T., GRAHAM, D.I., ADAMS, J.H., and FORD, I. (1989).Changes in the hippocampus and the cerebellum resultingfrom hypoxic insults: frequency and distribution. Acta Neu-ropathologica 78, 438^43.

381

NEWCOMB ET AL.

NILSSON, P., HILLERED, L., OLSSON, Y., SHEARDOWN,M.J., and HANSEN, A.J. (1993). Regional changes in inter-stitial K+ and Ca2+ levels following cortical compressioncontusion trauma in rats. J. Cereb. Blood Flow Metab. 13,185-192.

NIXON, R.A. (1986). Fodrin degredation by calcium-activatedneutral proteinase (CANP) in retinal ganglion cell neurons

and optic glia: Preferential localization of CANP activitiesin neurons. J. Neurosci. 6, 1264-1271.

NIXON, R.A., CLARKE, J.F., LOGVINENKO, K.B., TAN,M.K.H., HOULT, M., and GRYNSPAN, F. (1990). Alu-minium inhibits calpain mediated proteolysis and induces hu-man neurofilament proteins to form protein resistant highmolecular weight complexes. J. Neurochem. 55, 1950-1959.

PERLMUTTER, L.S., GALL, C, BAUDRY, M., and LYNCH,G. (1990). Distribution of calcium-activated protease calpainin the rat brain. J. Comp. Neurol. 296, 269-276.

PETTUS, E.H., CHRISTMAN, C.W., GIEBEL, M.L., andPOVLISHOCK, J.T. (1994). Traumatically induced alteredmembrane permeability: Its relationship to traumatically in-duced reactive axonal change. J. Neurotrauma 11, 507-521.

POSMANTUR, R., HAYES, R.L., DIXON, CE., and TAFT,W.C. (1994). Neurofilament 68 and neurofilament 200 pro-tein levels decrease after traumatic brain injury. J. Neuro-trauma 11, 533-545.

POSMANTUR, R.M., KAMPFL, A., LIU, S.J., HECK, K.,TAFT, W.C, CLIFTON, G.L., and HAYES, R.L. (1996).Cytoskeletal derangements of cortical neuronal processesthree hours after traumatic brain injury in rats: an immuno-fluorescence study. J. Neuropath. Exp. Neurol. 55, 68-80.

POSMANTUR, R.M., KAMPFL, A., SIMAN, R., LIU, S.J.,ZHAO, X., CLIFTON, G.L., and HAYES, R.L. (1997). Acalpain inhibitor attenuates cortical cytoskeletal protein lossafter experimental brain injury in the rat. Neuroscience, inpress.

POVLISHOCK, J.T., MARMAROU, A., McINTOSH, T.,TROJANOWSKI, J.Q., and MOROI, J. (1996). Impact ac-

celeration injury in the rat: evidence for focal axolemmalchange and related neurofilament sidearm alteration. J. Neu-ropath. Exp. Neurol., in press.

PULSINELLI, W.A., BRIERLEY, J.B., and PLUM, F. (1982).Temporal profile of neuronal damage in a model of transientforebrain ischemia. Ann. Neurol. 11, 491-498.

RAMI, A., and KRIEGLSTEIN, J. (1993). Protective effects ofcalpain inhibitors against neuronal damage caused by cyto-toxic hypoxia in vitro and ischemia in vivo. Brain Res. 609,67-70.

REGEHR, W.G., and TANK, D.W. (1994). Dendritic calciumdynamics. Curr. Opin. Neurobiol. 4, 373-382.

RIEDERER, B., and MATUS, A. (1985). Differential expres-sion of distinct microtubule-associated proteins during de-velopment. Proc. Nati. Acad. Sei. USA 82, 6006-6009.

ROBERTS-LEWIS, J.M., SAVAGE, M.J., MARCY, V.R.,PINSKER, L.R., and SIMAN, R. (1994). Immunolocalizationof p calpain mediated spectrin degradation to vulnerable neu-

rons in ischémie gerbil brain. J. Neurosci. 14, 3934-3944.

SAATMAN, K.E., BOZYCZKO-COYNE, D., MARCY, V.,SIMAN, R., and MCINTOSH, T.K. (1996a). Prolonged cal-pain-mediated spectrin breakdown occurs regionally follow-ing experimental brain injury in the rat. J. Neuropath. Exp.Neurol. 55, 850-860.

SAATMAN, K.E., MURAI, H., BARTUS, R.T., SMITH, D.H.,HAYWARD, N.J., PERRI, B.R., and MCINTOSH, T.K.(1996b). Calpain inhibitor AK295 attenuates motor and cog-nitive deficits following experimental brain injury in the rat.Proc. Nati. Acad. Sei. USA 93, 3428-3433.

SAIDO, T.C, SORIMACHI, H, and SUZUKI, K. (1994). Cal-pain: new perspectives in molecular diversity and physio-logical-pathological involvement. FASEB J. 8, 814-822.

SAIDO, T.C, YOKOTA, M., NAGAO, S., YAMAURA, L,TANI, E., TSUCHIYA, T., SUZUKI, K., andKAWASHIMA, S. (1993). Spatial resolution of fodrin pro-teolysis in postischemic brain. J. Biol. Chem. 268,25239-25243.

SAITO, K.I., ELCE, J.S., HAMOS, J.E., and NIXON, R.A.(1993). Widespread activation of calcium-activated neutralproteinase (calpain) in the brain in Alzheimer disease: a po-tential molecular basis for neuronal degeneration. Proc. Nati.Acad. Sei. USA 90, 2628-2632.

SCHLAEPFER, W.W., and ZIMMERMAN, U.J.P. (1985).Calcium-activated proteolysis of intermediate filaments.Ann. NY. Acad. Sei. 455, 552-562.

SHAPIRA, Y., YADID, G, COTEV, S., and SHOHAMI, E.(1989). Accumulation of calcium in the brain following headtrauma. Neurological Res. 11, 169-172.

SIESJOE, B.K., and BENGTSSON, F. (1989). Calcium fluxes,calcium antagonists, and calcium related pathology in brainischemia, hypoglycemia, and spreading depression: a unify-ing hypothesis. J. Cereb. Blood Flow Metab. 9, 127-140.

SIMAN, R., BAUDRY, M., and LYNCH, G. (1984). Brain fo-drin: Substrate for calpain I, an endogenous calcium-acti-vated protease. Proc. Nati. Acad. Sei. USA 81, 3572-3576.

SIMAN, R., NOSZEK, J.C, and KEGERISE, C. (1989). Cal-pain I activation is specifically related to excitatory aminoacid induction of hippocampal damage. J. Neurosci. 9,1579-1590.

STYS, P.K., SONTHEIMER, H., RANSOM, B.R., and WAX-MAN, S.G. (1993). Noninactivating, tetrodotoxin-sensitiveNa+ conductance in rat optic nerve axons. Proc. Nati. Acad.Sei. USA 90, 6976-6980.

SUZUKI, K., TSUJI, S., ISHIURA, S., KIMURA, Y., KUB-OTA, S., and IMAHORI, K. (1981). Autolysis of calcium-activated neutral protease of chicken skeletal muscle. J.Biochem. 90, 1787-1793.

382


TAFT, W.C., YANG, K, DIXON, CE., CLIFTON, G.L., andHAYES, R.L. (1993). Hypothermia attenuates the loss of hip-pocampal microtubule-associated protein 2 (MAP2) follow-ing traumatic brain injury. J. Cereb. Blood Flow Metab. 13,796-802.

TAFT, W.C., YANG, K, DIXON, CE., and HAYES, R.L.(1992). Microtubule associated protein 2 level in hippocam-pus following traumatic brain injury. J. Neurotrauma 9,281-290.

WANG, K.K.W., and YUEN, P.W. (1994). Calpain inhibition:an oveview of its therapeutic potential. TIPS 15, 412-419.

WHITSON, J.S., KAMPFL, A., ZHAO, X., and HAYES, R.L.(1995a). Time course of neurofilament protein loss follow-ing depolarization-induced injury in CNS culture. Neurosci.Lett. 197, 159-163.

WHITSON, J.S., KAMPFL, A., ZHAO, X., DIXON, CE., andHAYES, R.L. (1995b). Brief potassium depolarization de-creases levels of neurofilament proteins in CNS culture.Brain Res. 694, 213-222.

YAGHMAI, A., and POVLISHOCK, J.T. (1992). Traumati-cally induced reactive change as visualized through the use

of monoclonal antibodies targeted to neurofilament subunits.J. Neuropath. Exp. Neurol. 51, 158-176.

YAMAKAMI, I., and McINTOSH, T.K. (1991). Alterations inregional cerebral blood flow following brain injury in the rat.J. Cereb. Blood Flow Metab. 11, 655-660.

YOKOTA, M., SAIDO, T.C., TANI, E., KAWASHIMA, S.,and SUZUKI, K. (1995). Three distinct phases of fodrin pro-teolysis induced in postischemic hippocampus: Involvementof calpain and unidentified proteases. Stroke 26, 1901-1907.

YUSTE, R., GUTNICK, M.J., SAAR, D., DELANEY, K.R.,and TANK, D.W. (1994). Calcium accumulations in den-drites of neocortical pyramidal neurons: An apical band andevidence for two functional compartments. Neuron 13,23^13.

ZHAO, X., POSMANTUR, R.M., LIU, J.S., NEWCOMB, J.K.,CLIFTON, G.L., and HAYES, R.L. (1996). Time-related cal-pain I activity analysis following traumatic brain injury in rat

using casein zymography. Soc. Neurosci. Abstr 22, 1903.

ZIMMERMAN, U.J.P., and SCHLAEPFER, W.W. (1991).Two stage autolysis of the catalytic subunit initiates activa-tion of p calpain. Biochem. Biophys. Acta. 1078, 192-198.

Address reprint requests to:Ronald L. Hayes, Ph.D.

Vivian L. Smith Center for Neurologic ResearchDepartment of NeurosurgeryUniversity of Texas Houston

Health Science Center6431 Fannin Street, Suite 7.148

Houston, TX 77030

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