distinguishing excitotoxic from apoptotic neurodegeneration in the developing rat brain

Distinguishing Excitotoxic FromApoptotic Neurodegenerationin the Developing Rat Brain

M.J. ISHIMARU,1 C. IKONOMIDOU,2 T.I. TENKOVA,3 T.C. DER,3 K. DIKRANIAN,3

M.A. SESMA,3 AND J.W. OLNEY3*1Medical Research Institute, Tokyo Medical and Dental University, 2–3-10 Kanda-surugadai,

Chiyoda-ku, Tokyo, Japan2Deptartment of Pediatric Neurology, Charite, Virchow Clinics, Humboldt University,

13353 Berlin, Germany3Department of Psychiatry, Washington University School of Medicine,

St. Louis, Missouri 63110

ABSTRACTMuch confusion has arisen recently over the question of whether excitotoxic neuronal

degeneration can be considered an apoptotic phenomenon. Here, we addressed this questionby using ultrastructural methods and DNA fragmentation analysis to compare a prototypicapoptotic in vivo central nervous system cell death process (physiologic cell death in thedeveloping rat brain) with several central nervous system cell death processes in the in vivoinfant rat brain that are generally considered excitotoxic (degeneration of hypothalamicneurons after subcutaneous administration of glutamate and acute neurodegenerationinduced by hypoxia/ischemia or by concussive head trauma). We found by ultrastructuralanalysis that glutamate induces neurodegenerative changes in the hypothalamus that areidentical to acute changes induced in the infant rat brain by either hypoxia/ischemia or headtrauma, and that these changes are fundamentally different both in type and sequence fromthose associated with physiologic cell death (apoptosis). In addition, we show by ultrastruc-tural analysis that concussive head trauma induces both excitotoxic and apoptotic neurodegen-eration, the excitotoxic degeneration being very acute and localized to the impact site, and theapoptotic degeneration being delayed and occurring in regions distant from the impact site.Thus, in the head trauma model, excitotoxic and apoptotic degeneration can be distinguishednot only by ultrastructural criteria but by their temporal and spatial patterns of expression.Whereas ultrastructural analysis provided an unambiguous means of distinguishing betweenexcitotoxic and apoptotic neurodegeneration in each example analysed in this study, DNAfragmentation analysis (TUNEL staining or gel electrophoresis) was of no value because thesetests were positive for both processes. J. Comp. Neurol. 408:461–476, 1999. r 1999 Wiley-Liss, Inc.

Indexing terms: necrosis; glutamate; physiologic cell death; ischemia; head trauma

In the early 1970s, two new words, ‘‘apoptosis’’ and‘‘excitotoxicity,’’ were coined by two separate researchgroups who happened to be studying two apparentlydifferent types of cell death. Kerr and colleagues intro-duced the term apoptosis to refer to a cell death processthat had specific morphologic features and occurred in avariety of different circumstances, all having to do withcontrolled cell deletion (Kerr et al., 1972). These authors,on the basis of ultrastructural criteria, subsequently pro-posed that all cell death processes might fit into two broadcategories, which they named apoptosis and ‘‘necrosis’’(Wyllie et al., 1980). Olney and colleagues (Olney et al.,1971, 1974; Olney, 1974) introduced the term excitotoxicity

to refer to an acute process by which glutamate or itsvarious excitatory structural analogs trigger nerve celldeath in the central nervous system (CNS) of eitherrodents (Olney, 1971) or primates (Olney et al., 1972).

Grant sponsor: NIMH; Grant number: MH 38894; Grant sponsor: NIA;Grant number: AG 11355; Grant sponsor: NIDA; Grant number: DA 05072;Grant sponsor: NEI; Grant number: EY 08089; Grant sponsor: NARSAD.

*Correspondence to: John W. Olney, Department of Psychiatry, Washing-ton University School of Medicine, St. Louis, MO 63110.E-mail: [email protected]

Received 24 March 1998; Revised 24 November 1998; Accepted 14January 1999

THE JOURNAL OF COMPARATIVE NEUROLOGY 408:461–476 (1999)

r 1999 WILEY-LISS, INC.

Over the past decade, neuroscientists have becomeincreasingly interested in the role of apoptosis and genomi-cally programmed cell death processes in the CNS and inthe possibility that such processes may be relevant toneurodegenerative diseases. Concurrently, they have be-come interested in the role of excitotoxic mechanisms inneurodegenerative diseases, and recently have begun ad-dressing the question whether excitotoxic cell death (ECD)processes might be mediated by apoptotic mechanisms.Typically, consistent with the precedent set by Wyllie et al.(1980), neuroscientists have framed the issue in terms ofan ‘‘apoptosis vs. necrosis’’ dichotomy, and the findingshave been confusing; it has been reported by many labora-tories that excitotoxic neuronal degeneration, includingthat induced by excitotoxin agonists or ischemia, is anapoptotic process, and by others that it is a necrotic processor that it may be both apoptotic and necrotic.

Contributing to the confusion is the fact that althoughapoptosis was originally defined (Wyllie et al., 1980) inultrastructural terms, recent studies have relied heavilyon non-ultrastructural methods, primarily DNA fragmen-tation analysis, to diagnose apoptosis. Moreover, becausemuch of the apoptosis research has focused on non-CNStissues such as thymus and liver or on worms and flies orin vitro cultured cells, it does not provide optimal referencestandards for recognizing apoptosis in the in vivo mamma-lian CNS. Although Wyllie et al. did mention an example ofCNS apoptosis (physiologic cell death [PCD], the processby which redundant neurons are deleted from the develop-ing CNS), they provided no illustrations of this phenom-enon.

A major objective of the present study was to develop astage by stage ultrastructural characterization of PCD inthe infant rat brain and to compare this prototypic ex-ample of apoptosis with a prototypic ECD process, theprocess by which glutamate when injected subcutaneouslydestroys neurons in the hypothalamus of the infant ratbrain. We chose this ECD model because of the ease withwhich it can be manipulated for experimental purposesand because it has already been demonstrated by ultra-structural analysis that hypothalamic neurodegenerationinduced by glutamate is identical to the acute neurodegen-erative reaction induced in the infant rat brain by hypoxia/ischemia or concussive head trauma (Ikonomidou et al.,1989a,b, 1996a,b; Olney et al., 1989). However, this com-parative analysis has been performed only for the acutereaction induced by these conditions and may not hold truefor delayed or more slowly evolving degenerative reac-tions. In view of a recent report (Ikonomidou et al., 1996b)that head trauma in the infant rat brain causes both anacute and delayed neurodegenerative response and thatneurons degenerating by the delayed mechanism dis-played conspicuous evidence for internucleosomal DNAcleavage by the TUNEL (terminal deoxynucleotidyl trans-ferase-mediated dUTP-biotin nick end labeling) assay, weperformed an ultrastructural analysis of this delayedreaction and compared it with the ultrastructural appear-ance of PCD to determine whether it qualifies as a bonafide example of apoptosis. Although our primary emphasisin these experiments was on ultrastructural analysis, wealso analyzed both PCD and ECD processes by DNAfragmentation methods.

MATERALS AND METHODS

Animals

To study PCD, a total of nine litters of Sprague-Dawleyrat pups at 2, 4, or 7 days postpartum were used as isdescribed in the specific protocols below for combinedlight/electron microscopy, TUNEL staining, silver impreg-nation, or gel electrophoresis. All animal protocols con-formed to NIH guidelines and were approved by theWashington University Animal Care and Use Committee.Because neurons undergoing spontaneous apoptosis (PCD)were more abundantly present at 2 than at 4 or 7 dayspostpartum, we focused primarily on 2-day-old brains fordeveloping an ultrastructural stage-by-stage descriptionof the PCD process. On the other hand, because thedeveloping brain shows peak sensitivity to neurodegenera-tion induced by glutamate or head trauma on postnatalday 7, we used 7-day-old rats for studying these degenera-tive processes. To study excitotoxic cell death, 7-day-oldinfant rats were treated subcutaneously with either mono-sodium glutamate (2 gm/kg) or saline and were killed at 4,8, 16, or 24 hours after treatment and studied by theprotocols described below for light/electron microscopy,TUNEL staining, silver impregnation, activated microg-lia, or gel electrophoresis (n 5 six control and six experi-mental pups per time interval and per methodologicprocedure). To study delayed neurodegeneration associ-ated with head trauma, 7-day-old infant rats were sub-jected to concussive injury to the parietal cortex induced bya falling weight, as described below and previously (Ikono-midou et al., 1996a). The focus of the present study was ondelayed neurodegeneration that occurs in the cingulatecortex 24 hours after concussive trauma to the parietalcortex. Therefore, 24 hours after concussive head trauma,infant rats were killed and the cingulate cortex studied byprotocols described below for light/electron microscopy,TUNEL staining, or silver impregnation.

Method for producing traumatic brain injury

The device for delivering the impact injury consists of ahollow stainless-steel cylinder 40 cm in length, which isperforated at 1-cm intervals to prevent air compression inthe tube. The device, which is kept perpendicular to thetable top and to the surface of the skull, guides a fallingweight onto a footplate resting on the surface of the skull.The diameter of the footplate is 2 mm and is positioned sothat, before impact by the weight, it depresses the skullsurface 1.5 mm. A force of 165 g/cm2 produced by a 10-gweight is used to produce brain contusion. On the day ofthe trauma experiment, 7-day-old Sprague-Dawley ratpups were anesthetized with halothane and placed in amold fashioned to fit the contours of the skull and to holdthe skull in the desired attitude. After a skin incision wasmade to expose the skull surface, the center of the foot-plate was stereotaxically positioned 3 mm anterior and 2mm lateral to the lambda and was fixed in place so that itdepressed the skull 1.5 mm. The contusion impact wasdelivered unilaterally to the right side of the skull. Thisapproach produced cortical contusions of comparable sever-ity in all animals. Sham control animals were subjected toall of the same procedures except that the weight was notallowed to fall. Twenty-four hours after trauma or thesham procedure, animals were prepared for histologicevaluation of the brains by light and electron microscopymethods described below.

462 M.J. ISHIMARU ET AL.

Combined light and electron microscopy

Rat pups were deeply anesthetized with halothane andperfused transcardially with a fixative composed of 1.5%glutaraldehyde and 1% paraformaldehyde. The brainswere sliced transversely into 1-mm slabs, osmicated over-night (1% osmium tetroxide), dehydrated in graded etha-nols, cleared in toluene, and embedded flat in araldite.Thin sections, 1-µm thick, were cut by using glass knives(0.5-inch wide) on an MT-2B Sorval ultratome, heat driedon slides, and stained with Azure II and methylene blue forevaluation by light microscopy. To study glutamate-induced excitotoxic lesions, we cut sections through thearcuate nucleus of the hypothalamus at a level midwaybetween the rostral and caudal limits of the nucleus whereglutamate-induced damage is known to be most severe. Tostudy delayed apoptotic neurodegeneration in the cingu-late cortex of infant rats subjected to head trauma, sec-tions were cut through the cingulate cortex at a level A 2.9mm (Sherwood and Timiras, 1970) where a robust displayof apoptotic neurodegeneration consistently is observedafter trauma to the parietal cortex. Because our ultra-tomes are modified to accept special chucks that holdblocks containing entire transverse slabs of brain, and weuse wide glass knives that can cut a block face 0.5 incheswide 3 0.75 inches long, we were able to prepare thinplastic-embedded sections from the entire block face at anydesired brain level and used this approach to screenvarious brain regions for neurons undergoing physiologiccell death. This approach allows any given portion of anygiven brain to be evaluated by either light or electronmicroscopy. For electron microscopy, areas of special inter-est from a given block were trimmed to a smaller size,ultrathin sections were cut and suspended over a formvar-coated slot grid (1 3 2 mm opening), stained with uranylacetate and lead citrate, and viewed in a JEOL (Peabody,MA) 100C transmission electron microscope. Slot gridswere used because they permit a continuous viewing field(1 3 2 mm) uninterrupted by grid mesh bars.

TUNEL staining

Gavrieli et al. (1992) described a histologic method forstaining cells that have undergone DNA fragmentation byinternucleosomal endonuclease cleavage. This method isreferred to as the TUNEL method. To apply this method,the animals were anesthetized and perfused with 4%paraformaldehyde in 0.1 M phosphate buffered saline(PBS, pH 7.4) through the left cardiac ventricle. Thebrains were removed and cut on a Vibratome into 50-µmthick sections, which were incubated in a reaction mixture(supplied in Apop Tag In Situ Apoptosis Detection Kit,Oncor, Gaitersburg, MD) containing terminal deoxytrans-ferase, which incorporates nucleotides into the free 3’-OHterminals of DNA molecules. Because cells undergoingapoptosis have large numbers of DNA fragments with free3’-OH terminals, the transferase preferentially incorpo-rates nucleotides into these cells. The reaction mixturealso contains digoxigenin-11-dUTP (Boehringer Mann-heim, Indianapolis, IN), which enters into a terminaltransferase extension reaction. The incorporated digoxi-genin-dUTP was reacted with a horseradish peroxidaseconjugate of anti-digoxigenin antibody, and the antigen-antibody complex was detected by peroxidase reactionwith H2O2 and 3,3’-diaminobenzidine (DAB) as a chromo-gen. Sections were mounted on slides, optionally counter-

stained with methyl green and examined by light micros-copy.

Double labeling for glial fibrillary acidicprotein and TUNEL

Sections stained as above described by the TUNELmethod were thoroughly washed at room temperature in asolution of 0.01 M PBS/Triton X-100 (0.5%), then incu-bated for 1 hour in the same solution with 1% bovineserum albumin added. The sections were then incubatedovernight at 4°C in the presence of monoclonal antibodyagainst glial fibrillary acidic protein (GFAP; 1:400 dilu-tion; Sigma, St. Louis, MO). After PBS rinses, sectionswere incubated with goat anti-mouse BODIPY (1:200;Molecular Probes, Eugene, OR) in PBS/Triton X-100 for 1hour at room temperature. With a Kodak digital camera,microscopic fields were photographed under fluorescenceillumination to detect localization of the BODIPY fluores-cence probe and under ordinary illumination to detect theTUNEL DAB reaction product. The two images from agiven field were then superimposed in Adobe PhotoShop todetermine whether the GFAP and TUNEL labels werecolocalized in the same cells.

Silver impregnation

We have found that the DeOlmos cupric silver stainingmethod (DeOlmos and Ingram, 1971) is a useful methodfor marking degenerating neurons in the developing brainthat are dying by either an apoptotic or nonapoptoticmechanism. We use this method, in addition to the TUNELmethod, to determine whether the TUNEL method ismarking all of the cells that are dying and whether bothstains show the same pattern of distribution of dying cells.In the present study, we found that both methods showedthe same pattern of degeneration (physiologic cell death)when applied to the brains of saline-treated rats and bothshowed the same induced pattern of degeneration inspecific brain regions when applied to the brains of infantrats subjected to glutamate treatment or head trauma.

To visualize degenerating cells by the DeOlmos cupricsilver method, the brains were perfused with fixativecontaining paraformaldehyde (4%) in phosphate buffer, pH7.4, and serial transverse sections, 70µm thick, were cut byVibratome from the entire mid- and forebrain. Thesesections were stained with silver nitrate and cupric nitrateaccording to the protocol of DeOlmos and Ingram (1971).

Staining of activated microglia

It has been maintained that cells dying by necrosis elicita more robust inflammatory response than cells dying byapoptosis. It is known that the lesion induced in thehypothalamus by glutamate elicits a very rapid responseof cells presumed to be microglia. To study this reactionwith a more specific method than has been applied previ-ously, we used the cytochemical method for staining acti-vated microglia according to the protocol by Streit (1990).The rat pups were anesthetized and perfused with 4%paraformaldehyde in 0.1 M PBS through the left cardiacventricle. The brains were removed and cut on a Vibra-tome into 70-µm thick sections, which were washed inPBS, quenched in 2% H2O2, and blocked with 1% bovineserum albumen, and 0.1% Triton X-100 in PBS. Sectionswere then incubated in diluted (1:200) lectin-horseradishperoxidase conjugate complex (Griffonia simplicifolia seeds,GSA I-B4-HRP, obtained from Sigma) at room tempera-

APOPTOTIC VS. EXCITOTOXIC NEURODEGENERATION 463

ture for 2 hours. The lectin-binding sites were visualizedby reacting with H2O2 and DAB.

Gel electrophoresis

A characteristic feature of apoptotic cell death is thedegradation of genomic DNA by an endonuclease thatresults in internucleosomal cleavage of DNA into frag-ments that differ by 200 base pairs and, therefore, pro-duces a characteristic ‘‘laddering’’ pattern by agarose gelelectrophoresis. For this type of analysis, animals werekilled by decapitation and the brains removed quickly andfrozen immediately in powdered dry ice. Brain areas ofinterest were dissected from the brain on ice, homogenizedin 600 µl of DNA lysis solution consisting of 50 mMTris-HCl (pH 8.0), 10 mM EDTA (pH 8.0), 200 mM NaCl,0.5% SDS, and 0.4 mg/ml proteinase K at 60°C for 1 hour,and then incubated at 37°C overnight. Equal amounts ofphenol and chloroform were added to each sample, and themixture was centrifuged for 5 minutes. DNA samples werepipetted off as the water layer and were precipitated withan equal amount of isopropanol at -20°C for 1 hour. Theprecipitated pellet was washed in 70% ethanol, dried, andresuspended in 50 µl of Tris/EDTA buffer (TE). Sampleswere incubated with 1 mg/ml RNase A and incubated with0.4 mg/ml proteinase K at 37°C overnight. DNA wasextracted, precipitated, and suspended in 20 µl of TE. DNAwas labeled with digoxigenin-11-dd-UTP by TdT by usingGeniusy V system (Boehringer-Mannheim), then wasfractionated by electrophoresis in 1.8% agarose gel. TheDNA was transferred to a nylon membrane by capillaryaction, then was fixed by baking at 80°C for 3 hours. Themembrane was blocked in a 2% nucleic acid blockingsolution, then was incubated with anti-digoxigenin anti-body (1:10,000) conjugated to alkaline phosphatase, andthe labeled DNA was visualized by luminescence reactionby using CSPD (Boehringer-Mannheim). The nylon mem-brane was exposed to Kodak X-OMAT film for 5–10minutes.

Quantitative evaluation of trauma-inducedapoptotic neurodegeneration

All comparisons between traumatized and sham-trauma-tized animals were made on 8-day-old infant rats (24 hoursafter trauma or sham trauma on postnatal day 7). At thisage in the normal brain, physiologic cell death is occurringin many brain regions, but it is occurring at a very low rateso that in silver- or TUNEL-stained sections only anextremely low density of degenerating profiles can befound in any given brain region. However, 24 hours afterconcussive trauma to the parietal cortex, there is a markedincrease in the density of degenerating profiles in severalspecific brain regions, including the dorsal subiculum,laterodorsal thalamic nucleus, dorsomedial quadrant ofthe caudate nucleus, and layer II of the frontal, cingulate,and retrosplenial cortices. To provide a quantitative esti-mate of the increased rate of apoptotic neurodegenerationin these six brain regions after head trauma, we counteddegenerating (argyrophilic) cell bodies in sections stainedby the DeOlmos cupric silver method (DeOlmos and In-gram, 1971) by using an unbiased optical dissector ap-proach described by West (1993). The boundaries of theregions within which counts were made were consistentwith those delineated in the Sherwood and Timiras stereo-taxic atlas of the developing rat brain (1970). The frontalcortex and caudate nucleus were counted in sections cut at

A 4.7 mm, the cingulate cortex and laterodorsal thalamusat A 2.9 mm, and the retrosplenial cortex and subiculum atA 0.0 mm. The sampling was made by placing an unbiasedcounting frame (0.05 mm 3 0.05 mm; dissector height at0.07 mm) over each brain region, by using 8–10 randomlyselected dissectors and an objective lens with a highnumerical aperture. Because the DeOlmos silver methodstains both cell bodies and dendrites, only argyrophilicprofiles judged to be large enough to be cell bodies werecounted. The counts were made by an experienced histopa-thologist who was blind to the treatment condition. Fromthe counts made on traumatized (n 5 6) and sham-traumatized (n 5 6) brains, a mean density (6 SEM) ofdegenerating neuronal profiles was derived for each brainregion. Statistical significance for the comparison of meanvalues from each region of traumatized and sham-traumatized brains was evaluated by Student’s t-test.

RESULTS

Physiologic cell death

Light microscopic observations. In plastic thin sec-tions from untreated control animals PCD profiles weresometimes detectable as condensed shrunken cells withmultiple spherical chromatin masses, but often it wasdifficult to distinguish these cells from other relativelysmall, dark-staining cells that were not dying and were ofprobable glial origin.

In TUNEL-stained sections from normal control ani-mals, cells scattered widely throughout the brain withhigher concentrations being clustered in certain regionsdisplayed TUNEL positivity (Fig. 1). It is reasonable toassume that the TUNEL-positive cells are PCD cellsbecause there is no other explanation why they would beundergoing DNA degradation in the normal developingbrain. Therefore, the TUNEL stain provides a usefulmeans of studying the distribution pattern of cells undergo-ing physiologic cell death at any given time during develop-ment. In general, TUNEL-positive cells tended to be moreheavily concentrated in the vicinity of the cerebral ven-tricles and aqueduct of Sylvius (Fig. 1A) and sometimesappeared in a laminar display, apparently reflecting migra-tion patterns. In many of the cells that were TUNELpositive, the stain was not restricted to the nucleus, butrather was also distributed diffusely throughout the cyto-plasm. Many of the PCD profiles were relatively small andspherical with sparse processes. However, in some brainregions where larger neurons are ordinarily located, forexample, in the cerebral cortex, hippocampus, and deepcerebellar nuclei, TUNEL-positive profiles were seen thatwere large and conformed to the shape of moderatelyshrunken pyramidal or multipolar neurons (Fig. 1D). Inthese cases, the entire cell body, including the mainstemdendrite, was lightly TUNEL positive and the nucleus (ornuclear chromatin masses) was more prominently stained.In the arcuate nucleus of the hypothalamus of normalcontrol animals an occasional TUNEL-positive profile wasobserved (Fig. 1E).

We were not able to study microglial activation as aresponse to physiologic cell death because of the lack of anyage-matched control material for comparison, i.e., allbrains at a specific age in the normal brain showed thesame pattern of PCD and same pattern of microglial


activation in that these are phenomena occurring natu-rally in the normal developing brain for which there is nonegative control against which it can be compared.

Ultrastructural observations. We were able to studyby electron microscopy the apoptosis process in PCDneurons by first determining in TUNEL-stained sectionswhere these cells tend to be concentrated. Because a veryhigh concentration of TUNEL-positive cells was consis-tently present in regions dorsal and lateral to the aqueductof Sylvius, we performed our ultrastructural analysisprimarily in this brain region. The first detectable ultra-structural changes in cells undergoing PCD consisted ofclumping of nuclear chromatin and mild to moderatecondensation of the entire cell. It was not possible todetermine which of these changes occurred first, because

in the earliest detectable stages, there was evidence forboth of these changes. The most conspicuous early changewas in nuclear chromatin, which was transformed from apattern of homogeneously dispersed fine particulate mate-rial to larger caliber, flocculent densities in the midst ofwhich appeared abnormally large electron-dense chroma-tin masses (Fig. 2A) . These masses assumed a sphericalshape and appeared to grow in circumference by attach-ment of the flocculent densities to the outer surface of thesphere. Thus, in the earliest stages while incorporatingflocculent densities, they had a roughly contoured outline(Fig. 2A), which in later stages became more uniformlysmooth (Fig. 2B). In the earliest stages, cytoplasmic organ-elles did not appear to be undergoing a significant degen-erative process (Fig. 2A), although in later stages mitochon-

Fig. 1. Physiologic cell death as evidenced by TUNEL (terminaldeoxynucleotidyl transferase-mediated dUTP-biotin nick end label-ing) staining. A: TUNEL-stained Vibratome section through theaqueduct of Sylvius (AqS) at the level of the superior colliculusshowing a high concentration of TUNEL-positive profiles lateral anddorsal to the aqueduct in a 2-day-old rat pup. B: Vibratome sectionfrom the cerebellum of a 2-day-old rat pup showing relatively abun-dant TUNEL-positive profiles distributed in a scattered pattern incortical (cx) regions and regions containing deep (d) cerebellar nuclei.C: Vibratome section from the forebrain of a 2-day-old rat pup showing

TUNEL-positive cells distributed sparsely in the hippocampus (HC),corpus callosum (CC), and cingulate (Cn) cortex. D: Pyramidal neuronfrom the parietal cortex of a 2-day-old rat pup showing TUNELpositivity diffusely throughout the cell body and apical dendrite.Several dense chromatin balls are evident in the nuclear region.E: Vibratome section through the arcuate nucleus of the hypothala-mus of a 7-day-old rat pup showing an occasional TUNEL-positiveprofile (arrowheads). V3, third ventricle. Scale bars 5 100 µm in A–C,10 µm in D,E.


dria displayed a mild degree of swelling and disruption ofmembrane integrity (Fig. 3A). In the relatively earlystages, the nuclear envelope separated into fragments thatfloated randomly about the cytoplasm (Fig. 2B). Thefragments retained a bilaminar composition and appearedto be normal segments of nuclear membrane that hadbecome disconnected from one another. In the absence ofan intact continuous nuclear membrane, the cell becameunpartitioned with nucleoplasmic contents freely intermin-gling with cytoplasmic contents (Figs. 2B, 3A,B). Floccu-lent densities that originated in the nucleus becamediffusely distributed throughout the cell, and the largerchromatin masses migrated often toward the periphery ofthe cell (Figs. 2B, 3A). In some cases, the cell divided intoseparate independent bodies consisting of a contingent ofcytoplasm and (sometimes) one or more nuclear chromatinballs. These bodies sometimes appeared to separate fromthe main cell mass by a pinching off process (Fig. 3A). Inthe neuropil, many villous-like processes abutted uponthese bodies, but the bodies did not appear to be ingestedby or become engulfed within the cytoplasm of a phago-cyte. Rather, they appeared to remain free in the neuropilas they underwent biological degradation (Fig. 3B).

Agarose gel electrophoretic analysis. Electropho-retic analysis of DNA extracts from regions where rela-tively large numbers of PCD profiles were evident byTUNEL staining showed a laddering pattern (Fig. 4,lane 5).

Excitotoxin-induced cell death

Light microscopic observations. In plastic thin sec-tions, within 1 hour after subcutaneous administration ofglutamate, vacuous swelling of numerous processes in theneuropil gave a spongiform appearance to the arcuatenucleus of the hypothalamus. On a slightly more delayedschedule, cell bodies of arcuate hypothalamic neuronsbegan to show dramatic changes consisting of edematousswelling of the cytoplasmic compartment followed byclumping of nuclear chromatin and pyknosis of the nucleus.Cells undergoing these changes are known in the excitotox-icity literature as ‘‘bull’s eye’’ profiles. These changestranspired rapidly, so that by 4 hours after glutamateadministration, most of the affected arcuate neurons wereprominently showing such changes (Fig. 5). The dead ordying cells continued to be conspicuously evident as bull’s

Fig. 2. Ultrastructural depiction of early stages of physiologic celldeath (PCD) in the periaqueductal brain region of a normal 2-day-oldrat pup. In each figure, an inset shows an entire cellular profile at lowmagnification, and the figure itself presents portions of the cell athigher magnification. A: This neuron shows the earliest signs of PCDconsisting of mildly condensed cytoplasm, flocculent densities appear-ing throughout the nucleus, and dense chromatin masses forming inthe nucleus apparently by incorporation of the flocculent densities.Note that, at this early stage, relatively normal mitochondria (m) arepresent in the cytoplasm, the nuclear membrane is relatively intactand the nucleoplasmic contents are still confined largely within the

nucleus. B: This neuron shows a slightly later stage of PCD featuring aseparation of the nuclear envelope into discontinuous linear frag-ments that cease enclosing the nucleus, thereby allowing intermixingof nucleoplasmic and cytoplasmic contents. The flocculent densitiesthat originated in the nucleus are dispersed throughout the cyto-plasm, and a large dense smoothly contoured chromatin ball hasmigrated to the periphery of the cell. The bilaminar strands of nuclearmembrane curl upon themselves (arrowheads) and appear to enclose amass of intermixed cytoplasm and nucleoplasm. Scale bars 5 2 µm inA,B, 10 µm in insets.


Fig. 3. Ultrastructural depiction of more advanced stages of physi-ologic cell death (PCD) in the periaqueductal brain region of a normal2-day-old rat pup. A: The neuron in the inset, detailed at highermagnification in the figure, appears to be in the process of forming anapoptotic body. It displays two dense chromatin balls, one of which hasmigrated to the periphery where it is on the leading edge of aprotuberance that appears to be undergoing a pinching off process,possibly with the assistance of a lip of cytoplasm (arrowhead) from

another cell that is insinuating itself at the neck of the protuberance.In this stage, mitochondria (m) typically show mild swelling. B: Thisscene depicts an apoptotic cell surrounded by debris that may beapoptotic bodies undergoing biological degradation in the neuropil.These masses of debris are frequently found adjacent to cells undergo-ing PCD and are typically surrounded by numerous fine-caliberprocesses but are not incorporated into a phagocytic cell. Scale bars 52 µm in A, 15 µm in inset, 1.5 µm in B.


eye profiles at 8 hours after treatment, but at 16 and 24hours, they were detectable only as small dark structures(Fig. 5).

Four hours after glutamate treatment, although degen-erating arcuate neurons showed conspicuous clumping ofnuclear chromatin in plastic thin sections, the TUNELstain applied to Vibratome sections to detect internucleo-somal cleavage was negative (Fig. 5) except for a rarepositively stained profile that could be interpreted as aneuron undergoing PCD, because control sections fromnormal saline-treated or untreated animals also showedan occasional TUNEL-positive profile in the arcuate hypo-thalamic region (Fig. 1E). However, at 8 hours, manyTUNEL-positive profiles could be seen in the arcuateregion, and even larger numbers were evident at 16 hours(Fig. 5). Between 16 and 24 hours, the number of TUNEL-positive profiles declined. At each time interval whenTUNEL staining was evident, the stain was not restrictedto the nucleus but rather was diffusely distributed through-out the cell. In addition, it sometimes appeared as if theTUNEL positivity was not confined to the degeneratingneuron but might be present in a larger complex formed bythe degenerating neuron and surrounding phagocytic cells.

In Vibratome sections processed cytochemically to showmicroglial activity, such activity was minimally present inthe arcuate hypothalamic region at 0 or 4 hours afterglutamate treatment but became clearly detectable at 8hours, reached a peak at 16 hours, and diminished by 24hours (Fig. 5).

Ultrastructural observations. By electron micros-copy, it was evident that the early spongiform changes inthe neuropil consisted of massive dilation of dendriticprocesses (Fig. 6A) . Axonal processes coursing through orterminating in the region did not show pathologic changes.In the cytoplasm of arcuate neurons, the changes occurredin a characteristic sequence, beginning with vacuolation ofendoplasmic reticulum. This process was accompanied bycondensation and shrinkage of mitochondria (Fig. 6B).However, in a slightly later period, but still within the first2 hours, mitochondria began to swell and became progres-sively more edematous, swollen, and vacuous. At the sametime that these changes were occurring, the rough endo-plasmic reticulum disintegrated with polyribosomes becom-ing disaggregated and ribosomal particles becoming scat-tered diffusely throughout the cytoplasm. Nuclear changesdid not occur until the cytoplasmic changes were alreadyrelatively well developed. The earliest changes in thenucleus became evident at approximately 2 hours aftertreatment. They consisted of nuclear chromatin formingnumerous small clumps with individual clumps migratingfirst to the nuclear envelope to form a clock-face profile,then coalescing centrally into a single electron-densepyknotic mass having irregular borders (Fig. 6C–F). Al-though the nuclear limiting membranes became crenu-lated, at no time throughout the degenerative process didthese membranes disintegrate or cease enclosing thenucleus in a continuous manner, and at no time did thenucleoplasmic and cytoplasmic contents show signs ofintermixing (Fig. 6C–G). In the 4- to 8-hour interval,phagocytic cells wrapped pseudopodic processes aroundthe degenerated cell (Fig. 6G,H), engulfing it in toto, andthe degenerated cell was biologically degraded within thecytoplasm of the phagocyte or was disgorged as partiallydigested debris into the vascular system.

Because the presence of TUNEL-positive profiles in thearcuate nucleus at 8, 16, and 24 hours suggests thepossibility that glutamate treatment triggers two types ofneurodegeneration, an excitotoxic process in the acute4- to 6-hour interval followed by a delayed wave ofapoptotic neurodegeneration in the 8- to 24-hour interval,we examined the degenerating profiles very carefully at 8,16, and 24 hours in search for evidence of an apoptoticneurodegenerative process. No such evidence was found.The degenerating profiles in the arcuate nucleus at thesepost-treatment intervals consistently displayed ultrastruc-tural characteristics of a late-stage excitotoxic process,which in most cases was occurring within the cytoplasmicconfines of phagocytic cells.

Agarose gel electrophoretic analysis. Electropho-retic analysis of DNA extracts from the arcuate hypotha-lamic region of glutamate-treated animals showed a ladder-ing pattern, most prominent at 8 and 16 hours, which isconsistent with the observation that TUNEL positivitywas also most prominent at 8 and 16 hours after glutamatetreatment (Fig. 4).

Acute neurodegeneration induced byhypoxia/ischemia or concussive head trauma

In prior studies, the neurodegenerative process inducedin the infant rat hypothalamus by glutamate has beencompared with the acute neurodegenerative response tohypoxia/ischemia or concussive head trauma and the threeprocesses were found, by either light or electron micro-scopic analysis, to be identical. For documentation of these

Fig. 4. DNA fragmentation studied by gel electrophoresis. Thetissue extracts in lanes 1, 2, 3, and 4 are from the arcuate nucleus ofthe hypothalamus of 7-day-old rat pups 4, 8, 16, and 24 hours,respectively, after glutamate treatment. A laddering pattern is faintlyevident at 4 hours, becomes more prominent at 8 and 16 hours, andtapers off at 24 hours. The tissue extract in lane 5 from the periaque-ductal region of a normal untreated 2-day-old rat pup shows aladdering pattern as would be expected based on the high concentra-tion of TUNEL-positive cells undergoing physiologic cell death in thisbrain region (Fig. 1A). However, because the arcuate hypothalamicnucleus of the 7-day-old rat pup does not normally contain a highconcentration of cells undergoing physiologic cell death (Fig. 1E), theladdering pattern shown in lanes 1–4 reflects internucleosomal DNAcleavage associated with excitotoxic degeneration of arcuate neurons.


findings, please see the extensive illustrations in Olney(1971), Ikonomidou et al., (1989a), and Ikonomidou et al.(1996a).

Delayed neurodegeneration inducedby concussive head trauma

Light microscopic observations. In either silver-impregnated or TUNEL-stained sections, degeneratingneurons stained prominently against a light backgroundand could be detected in both the traumatized and sham-traumatized brains. By casual inspection, it was apparentthat the concentration of degenerating cells was increased

in several regions of the traumatized brains, including thedorsal subiculum, laterodorsal thalamic nucleus, dorsome-dial quadrant of the caudate nucleus, and layer II of thefrontal, cingulate, and retrosplenial cortices. In Figure 7, aquantitative estimate of the density of degenerating neuro-nal profiles in these six brain regions after concussivetrauma is compared with density counts for the same sixregions after sham trauma. In each of these brain regions,there was a marked increase in cell degeneration associ-ated with trauma, the magnitude of the increase varyingfrom 6-fold in the frontal cortex to 130-fold in the subicu-lum. The appearance of degenerating neurons in the

Fig. 5. Excitotoxic neurodegeneration in the arcuate hypothalamicnucleus of the 7-day-old infant rat at 4, 8, 16, or 24 hours aftersubcutaneous treatment with glutamate. In column I, the hypothala-mus is plastic embedded and stained with methylene blue/Azure II toshow neurons undergoing excitotoxic degeneration (bull’s eye profiles).In column II, the brain is Vibratome sectioned, exposed to TUNELreagents, and counterstained with methyl green. The profiles stainedmedium to dark brown are degenerating neurons showing TUNELpositivity. In column III, the brain is Vibratome sectioned and stained

cytochemically to show activated microglia (dark spider-like profiles)and vascular endothelium (amber tubular structures). Note that at 4hours after glutamate treatment, neuronal degeneration, includingnuclear pyknosis, is already evident by routine histology (column I),but the degenerating neurons do not become TUNEL positive (columnII) until 8 hours and show peak TUNEL positivity at 16 hours. Themicroglial response (column III) is in synchrony with the TUNELresponse, being first detected at 8 hours and becoming most prominentat 16 hours. Scale bar 5 20 µm (applies to all panels).


Fig. 6. Ultrastructural evolution of glutamate-induced excitotoxicneurodegeneration in the arcuate hypothalamic nucleus of the 7-day-old infant rat. A: The earliest detectable changes are in dendrites (D)of arcuate neurons, which show edematous swelling, whereas the axon(A) terminals in presynaptic contact with these dendrites retain anormal appearance. Mitochondria (m) in the dendrites rapidly becomemassively swollen. B: The earliest cytoplasmic changes in the cell bodyregion are condensation and thickening of mitochondrial (m) mem-branes and vacuole formation by saccules of endoplasmic reticulum(er). The early condensation change in mitochondria is very transientand is followed by a stage of vacuous swelling (C,D) and then a finalstage in which the mitochondrial matrix becomes moderately denseand the mitochondrion assumes a perfectly spherical shape (E–G). Thesequence of nuclear changes is depicted in C–F. C: Although mitochon-

dria are severely swollen throughout the cytoplasm, nuclear changesare only equivocally evident. D: A moderately more advanced stageshows conspicuous clumps of nuclear chromatin forming at the marginof the nucleus in a clock-face pattern. E,F: These clumps progressivelycoalesce and move to the center of the nucleus as a large irregularelectron-dense mass. G: An early stage of phagocytosis is depicted inwhich an entire cell, including the nucleus (n) and cytoplasm (cp), isengulfed by a glial (g) process that has incorporated the degeneratingcell into its cytoplasmic compartment. H: A more advanced stage ofphagocytosis is shown in which the nucleus of a glial cell (gn) is closelyapposed to two necrotic neurons that the glial cell has ingested in toto.A rim of glial cytoplasm completely surrounds and encloses thesenecrotic neurons. Scale bars 5 1 µm in A–H.


cingulate cortex 24 hours after head trauma is depicted bysilver staining in Figure 8A or by TUNEL in Figure 8B forcomparison with a TUNEL-stained section from a shamcontrol in Figure 8C.

Ultrastructural observations. Electron microscopicevaluation of the degenerating cells in the cingulate cortex24 hours after head trauma revealed that these cells wereundergoing changes indistinguishable from the PCD pro-cess described above and illustrated in Figures 2 and 3.The earliest change consisted of the formation of electron-dense chromatin balls in the nucleus, which was accompa-nied by a nuclear membrane beginning to show fragmenta-tion (Fig. 8D). In later stages, the nuclear membranebecame totally fragmented as the cell underwent condensa-tion and there was extensive intermixing of nucleoplasmicand cytoplasmic contents (Fig. 8E). These were the changesconsistently observed in large numbers of degeneratingcingulate cortical cells studied (n $ 100 degeneratingcells), and none of these cells showed signs of excitotoxicdegeneration. In late stages of degeneration, these cellswere decomposed beyond recognition, but in early stages,they retained morphologic characteristics, including theirsize, shape, and ultrastructural appearance identifyingthem most likely as neurons.

Double-label (GFAP and TUNEL) evaluation. Thedouble-label approach applied to the cingulate cortexrevealed that cells positive for GFAP were a separatepopulation from those positive for TUNEL (Fig. 9). Be-cause the TUNEL-positive degenerating profiles were not

GFAP positive, were often too large to be oligodendrocytesor microglia, and by electron microscopy had ultrastruc-tural characteristics more consistent with neurons thanglia, it is reasonable to conclude that the cells undergoingdelayed degeneration after head trauma are neurons.

DISCUSSION

Wyllie and colleagues (1980) described apoptosis as aprocess beginning with two conspicuous changes: theformation of electron dense chromatin masses in thenucleus and condensation of the entire cell. These changesoccurred in the absence of conspicuous changes in cytoplas-mic organelle systems and were followed by disintegrationof the nuclear membrane, migration of nuclear chromatinmasses into the cytoplasm, and protuberances separatingfrom the cell to form ‘‘apoptotic’’ bodies. Our findingsindicate that PCD, a cell death process that occurs sponta-neously in the developing CNS, mirrors the Wyllie et al.morphologic description of apoptosis. In contrast, theexcitotoxic cell death (ECD) process induced in the infantrat hypothalamus by systemically administered gluta-mate does not resemble this description. The earliestchanges associated with the ECD process consisted ofmassive edematous swelling of neuronal dendrites and cellbodies with accompanying changes in cytoplasmic organ-elle systems. Striking changes in nuclear chromatin oc-curred but not until after cytoplasmic organelle systemshad already undergone conspicuous disintegrative changes.The nuclear envelope assumed a crenulated appearancebut remained intact, and there was no evidence of nuclearchromatin material becoming dispersed into the cytoplasmor of the cell dividing into separate ‘‘bodies’’ containing amixture of nuclear and cytoplasmic material.

Previously, we have shown (Ikonomidou et al., 1989a)that glutamate-induced neurodegeneration (ECD) in theinfant rat hypothalamus is ultrastructurally identical toacute ischemia-induced neurodegeneration in several re-gions of the infant rat brain; here, we show by ultrastruc-tural criteria that ECD is not apoptotic, from which itfollows that acute ischemia-induced neurodegeneration ininfant rat brain is not apoptotic. This conclusion appearsto be at odds with several recent studies (Kure et al., 1991;Tominaga et al., 1993; Linnik et al., 1993; MacManus etal., 1993, 1994; Filipkowski et al., 1994; Sei et al., 1994;Beilharz et al., 1995; Bonfoco et al., 1995; Ferrer et al.,1995) in which cell death induced by an excitotoxin agonist(glutamate, kainic acid, N-methyl-D-aspartate [NMDA]) orischemia has been interpreted as an apoptotic or apoptosis-like process. In these studies, the apoptosis diagnosis wasentertained on the basis of light microscopic evidence forconspicuous clumping of nuclear chromatin and/or a posi-tive internucleosomal DNA cleavage reaction. Here, weshow that either apoptosis or an excitotoxic stimuluscauses conspicuous clumping of nuclear chromatin; but, byelectron microscopy, the chromatin changes associatedwith apoptosis can be distinguished from those triggeredby an excitotoxic stimulus on the basis of both ultrastruc-tural appearance and time sequence. If the morphologicchanges are fundamentally different by ultrastructuralappearance and time sequence, the two processes cannotbe considered the same, and the fact that tests for inter-nucleosomal DNA cleavage are positive for both processesdoes not signify that both processes are apoptotic; itsignifies that internucleosomal DNA cleavage tests are not

Fig. 7. Numerical density of degenerating neurons was estimatedby an optical dissector method in silver-stained sections from six brainregions 24 hours after trauma or sham trauma to the parietal cortex of7-day-old infant rats. The low density of degenerating neurons presentin each brain region of the sham controls represents the rate ofspontaneous apoptosis. In each region of the traumatized brains, thedensity counts were significantly (P , 0.001; Student’s t test) higherthan the spontaneous rate. Fr, frontal cortex, layer II; Cing, cingulatecortex, layer II; RS, retrosplenial cortex, layer II; Caud, caudatenucleus, dorsomedial quadrant; LD-Th, laterodorsal nucleus of thala-mus; Subic, dorsal subiculum.


Fig. 8. These photomicrographs illustrate trauma-induced distantapoptotic neurodegeneration in the posterior cingulate cortex of the8-day-old rat brain 24 hours after concussive injury to the parietalregion. A: The histologic section is stained by the DeOlmos cupricsilver method, which shows a dense pattern of degeneration primarilyaffecting neurons in layers II and V & VI, but with degeneratingelements in other layers as well. Note that the degeneration isrestricted to the right side, which is ipsilateral to the trauma. B,C: Thesections are stained by the TUNEL method; B is from a traumatizedbrain, and C is from an untraumatized control brain. The lesion in B isnot as visually striking as in A, primarily because the DeOlmos silver

method stains both the cell body and its processes, whereas theTUNEL stain is primarily restricted to the cell body region. D,E: Theultrastructural appearance of cingulate neurons undergoing delayedtrauma-induced degeneration. The neuron in D shows early apoptoticchanges primarily consisting of spherical balls of clumped chromatinand discontinuity of the nuclear membrane (arrow heads). The neuronin E shows more advanced changes, including a totally fragmentednuclear membrane, intermixing of nucleoplasmic and cytoplasmicelements and condensation of the entire cell. Scale bars 5 50 µm inA–C, 1 µm for D,E.


specific for apoptosis and are not valid for distinguishingapoptotic from other forms of cell death. This conclusion isconsistent with findings from several other laboratories(Collins et al., 1992; Grasl-Kraupp et al., 1995; Charriaut-Marlangue and Ben-Ari 1995; Gwag et al., 1997).

Ikonomidou et al. (1996a,b) have shown that concussivetrauma to the parietal cortex of the infant rat causesneurons at the local impact site to degenerate rapidly(within 4–6 hours) by an excitotoxic mechanism, and otherneurons at distant sites to degenerate more slowly over a

24-hour period. The distant, delayed cell death processwas considered apoptotic based on TUNEL positivity andpreliminary morphologic examination. In the present study,we evaluated in detail the ultrastructural appearance ofneurons undergoing delayed degeneration in the cingulatecortex after parietal cortical trauma and found that thisdegenerative reaction is identical to the PCD processdescribed herein and bears no resemblance to excitotoxicdegeneration. The observation that head trauma cantrigger two types of brain damage, one having ECD andthe other PCD characteristics, and that each type ofdamage has its own time schedule and distribution pat-tern is consistent with the interpretation that ECD andPCD are fundamentally different types of cell death pro-cesses. Portera-Cailliau et al. (1997) recently describedexcitotoxic and apoptotic degeneration occurring at thesame lesion site after kainic acid injection into the infantrat cerebral cortex and advanced the hypothesis that theexcitotoxic stimulus caused neurons to die by a hybrid‘‘continuum’’ process beginning with necrotic changes andending with apoptotic changes. Our findings suggest adifferent interpretation, i.e., that excitotoxic cell death hasits own unique course from beginning to end, and apoptoticcell death has its own unique course from beginning to end.We have found no evidence suggesting that an excitotoxicstimulus directly triggers an apoptotic response; a morelikely interpretation of the observations of Portera-Cailliau et al. (1997) would be that injection of kainic acidinto the immature cortex acutely kills many neurons by adirect excitotoxic mechanism, and causes other neurons toundergo apoptotic degeneration based on a separate mech-anism involving activation of a death program secondaryto excitotoxic destruction of synaptic targets and/or grossdisruption of the nutrient environment that these develop-ing neurons depend on for survival. This mechanismwould explain the presence of cells showing excitotoxicchanges side by side with cells showing apoptotic changes,but would not justify the interpretation that a given cellunderwent a sequence of early excitotoxic changes thatsubsequently evolved into apoptotic changes.

To avoid potential confusion in applying the criteriadescribed here for distinguishing apoptotic from excito-toxic cell death, it is necessary to point out that the seriesof morphologic changes described here for glutamate-induced ECD degeneration is only one of several patternsof morphologic changes that are triggered by excitotoxicstimulation. In recent publications (Ikonomidou et al.,1996c; Olney and Ishimaru, 1999; Corso et al., 1997), wehave described three distinct patterns of excitotoxic neuro-nal degeneration, none of which ultrastructurally meetcriteria for apoptosis. A major reason for the differentresponse patterns is that different CNS neuronal cell typesrespond differently to excitotoxic stimulation. We haveobserved that this difference is true whether the stimulusis from an exogenous excitotoxin agonist or from endog-enous glutamate under ischemic or head trauma condi-tions (Ikonomidou et al., 1989a,b, 1996a,c). Therefore,when attempting to distinguish by ultrastructural analy-sis between excitotoxic and apoptotic processes, it isimportant to remember that excitotoxicity has severaldifferent patterns of morphologic expression.

Although we have focused here on PCD, a cell deathprocess that is universally recognized as an example ofapoptosis, and we have shown that this process in thedeveloping rat brain closely resembles the examples of

Fig. 9. Vibratome sections were stained by the TUNEL methodand also reacted immunocytochemically with fluorescently taggedantibodies to glial fibrillary acidic protein (GFAP). The GFAP reactionproduct (bright green by color photography and light in black andwhite photography) depicts the presence of astroglial cell bodies andtheir fibrous processes in the cingulate cortex subjacent to the corticalsurface. In the same microscopic field, there are TUNEL-positive cells(dark spherical structures) that are GFAP negative, and this ischaracteristic of the staining pattern throughout all regions of thetraumatized brains. No evidence for colocalization of TUNEL andGFAP positivity in the same cells was observed in any brain region,including both gray and white matter regions. Scale bar 5 10 µm.


apoptosis described by Wyllie et al. (1980) in variousnon-CNS cells, it remains to be determined whetherapoptotic neurodegeneration in the mammalian CNS hasonly one basic morphologic form of expression. Our descrip-tion of the sequence of changes that characterize the PCDprocess is based primarily on studying neurons in themidbrain of the postnatal day 2 rat. However, we also haveexamined neurons undergoing PCD in the caudate nucleus,parietal cortex, and dorsolateral nucleus of the thalamusof the postnatal day 2 rat and have found that ultrastruc-turally these neurons show the same changes as thoseundergoing PCD in the midbrain. Moreover, we havedemonstrated here that pyramidal neurons undergoingdelayed degeneration in the cingulate cortex of the 7-day-old rat after concussive head trauma show the samechanges as those undergoing PCD in the midbrain onpostnatal day 2. In addition, we have recently discovered(Ikonomidou et al., 1998, 1999) that pharmacologic block-ade of NMDA glutamate receptors triggers a robust waveof apoptotic neurodegeneration, deleting millions of neu-rons from several major regions of the developing ratbrain, and in each region, the degenerative process isidentical to the PCD process described herein (Dikranianet al., 1998). Therefore, there appears to be a high degreeof consistency in the morphologic appearance of neurons invarious regions of the developing rat brain that areundergoing either spontaneous apoptosis or apoptosisinduced by head trauma or by blockade of NMDA recep-tors.

In apparent contrast to our findings, Clarke (1990) haspresented evidence that developmental cell death mayoccur by at least three different mechanisms, each havingits own pattern of morphologic expression, and only one ofthese patterns resembles the PCD process described herein.It is not clear whether there is a contradiction between ourfindings and those summarized by Clarke (1990) in thatthe cases cited by Clarke pertain primarily to nonmamma-lian species and/or involve experimental manipulationsdifferent from those performed in our experiments. Li et al.(1998) have shown that spinal motoneurons in immaturemice degenerate after peripheral nerve injury by a processthat the authors characterized as a mixture of apoptosisand necrosis, although the illustrations provided showvery few features resembling the PCD apoptotic processdescribed herein. Collectively, our findings and those ofClarke (1990) and Li et al. (1998) suggest the interpreta-tion that when cell death is triggered by an experimentalmanipulation, the type of cell death process will depend onthe type of manipulation. Comparing our findings (Ikono-midou et al., 1998a,b; Dikranian et al., 1998; the presentstudy) with those of Li et al. (1998), it appears thatperipheral nerve injury triggers a different cell deathprocess than is triggered by head trauma or blockade ofNMDA receptors. Although it seems reasonable to use theterm apoptosis or ‘‘programmed cell death’’ to refer to anycell death process (whether occurring spontaneously or byexperimental manipulation) that is ultrastructurally iden-tical to PCD, we question the practice of using such termsto refer to cell death processes that are triggered byexperimental manipulation and do not resemble PCD.

It has been claimed in three prior reports (Rink et al.,1995; Colicos et al., 1996; Pravdenkova et al., 1996) thatbrain trauma can cause apoptotic neurodegeneration inthe adult rat brain. Although it would be of interest tocompare in a point by point manner our findings in the

developing CNS with these earlier findings, this compari-son is not possible because no ultrastructural evidence isprovided in the earlier studies except for a single electronmicrograph in the study of Rink et al., which depicts at lowmagnification an apparent apoptotic cellular profile in arelatively late stage of degeneration. Therefore, the impor-tant question of whether trauma induces an apoptoticneurodegenerative reaction in both the immature andmature CNS and whether the apoptotic process displayssimilar ultrastructural characteristics at both ages cannotbe addressed until a more adequate set of ultrastructuraldata on adults becomes available.

Regarding the question of whether ECD processes shouldbe classified as necrosis, we believe that they should.However, we would not frame the issue in terms of anapoptosis vs. necrosis dichotomy because necrosis is anall-inclusive generic term that, according to medical dictio-naries, means ‘‘deadness’’ or ‘‘death of a cell;’’ therefore,strictly speaking, the issue is being framed in terms of‘‘apoptosis vs. deadness,’’ whereas the intended framingwould be apoptosis vs. other forms of cell death. Inaddition, it may be overly simplistic to assume that all celldeath processes can be subdivided into two mutuallyexclusive categories that are diametric opposites and donot share a number of basic overlapping properties. More-over, we question the proposal by Wyllie et al. (1980) thattheir description of necrosis can fit all non-apoptotic celldeath processes. None of the several excitotoxic cell deathprocesses that we are familiar with fit very well into theirnecrosis definition. Wyllie et al. (1980) did not emphasizenuclear chromatin clumping as a conspicuous feature ofnecrosis, and they suggested that electron-dense clumps ofchromatin became progressively less conspicuous and dis-appeared in the later stages of the necrosis process.Glutamate-induced excitotoxic cell death certainly doesnot fit this description; rather, it involves the formation oflarge masses of clumped nuclear chromatin that becomeincreasingly more conspicuous as the cell death processevolves. Because it is possible to specify several excitotoxiccell death processes that, by ultrastructural analysis, aredifferent from one another, are different from apoptosisand are different from the Wyllie et al. (1980) definition ofnecrosis, we question the adequacy, applicability, andutility of the Wyllie et al. apoptosis vs. necrosis frame ofreference for studying cell death processes in the in vivomammalian CNS.

In studying the PCD process, we have been impressedthat it has its own gestalt (temporospatial patternedsequence), which is entirely different from that of the ECDprocess. The PCD process has the appearance of beinginitiated from within the nucleus and being enacted in aspecific sequence of orderly steps seemingly programmedto achieve the predetermined goal of terminating its ownexistence, after first disassembling itself and arranging forthe piecemeal disposal of its remains. None of the ECDprocesses we have examined appear to be initiated fromwithin and none have the appearance of being an orderlyself-disassembly process. Presumably, the reason for thisdifference is that the PCD process does represent theenactment of a programmed performance and the ECDprocesses do not, except insofar as the cell has a pro-grammed plan for reacting to accidental lethal injury. Wesuspect that nerve cells have many different plans forreacting to accidental injury, depending on the type of celland nature of the injury, and each of these responses may


have its own peculiarities that will be reflected as subtledifferences in the ultrastructural or biochemical characterof the response. Some such responses may be borrowedfrom or mimic the cell’s apoptosis repertoire (for example,cell condensation, chromatin clumping, degradation ofDNA into fragments of a specific size). In our opinion, thiswould not justify attaching an apoptosis label to anaccidental cell death process that lacks the characteristicapoptosis gestalt. The diagnosis depends not on just onestructural manifestation or another but on a complexseries of ultrastructurally characterizable changes thatoccur in a specific sequence.

Sloviter et al. (1993), on the basis of a careful ultrastruc-tural evaluation, have reported that adrenalectomy-induced degeneration of hippocampal dentate granuleneurons in adult rats is an apoptotic process. The ultra-structural changes described by these authors for anapoptotic process in the adult rat CNS are similar to thosewe describe here for PCD in the developing CNS. Untilbetter criteria are developed, we propose that these descrip-tions, one for the adult and the other for the developingCNS, can serve as reference standards for determiningwhether a given cell death process in the in vivo mamma-lian CNS qualifies for a diagnosis of apoptosis.

ACKNOWLEDGMENTS

Supported in part by NIA grant AG 11355, NIDA grantDA 05072, NEI grant EY 08089, and by an NIMH Re-search Scientist Award MH 38894 and NARSAD Estab-lished Investigator Award, both to J.W.O.

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distinguishing excitotoxic from apoptotic neurodegeneration in the developing rat brain

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