primary sensory and forebrain motor systems in the newborn brain are preferentially damaged by...

Primary Sensory and Forebrain MotorSystems in the Newborn Brain Are

Preferentially Damaged byHypoxia-Ischemia

LEE J. MARTIN,1,* ANSGAR BRAMBRINK,2 RAYMOND C. KOEHLER,2

AND RICHARD J. TRAYSTMAN2

1Departments of Neuroscience, Pathology, and the Neuropathology Laboratory, JohnsHopkins University School of Medicine, Baltimore, MD 21205-2196

2Anesthesiology and Critical Care Medicine, Johns Hopkins University School of Medicine,Baltimore, Maryland 21205-2196

ABSTRACTCerebral hypoxia-ischemia causes encephalopathy and neurologic disabilities in new-

borns by unclear mechanisms. We tested the hypothesis that hypoxia-ischemia causes braindamage in newborns that is system-preferential and related to regional oxidative metabolism.One-week-old piglets were subjected to 30 minutes of hypoxia and then seven minutes ofairway occlusion, producing asphyxic cardiac arrest, followed by cardiopulmonary resuscita-tion and four-day recovery. Brain injury in hypoxic-ischemic piglets (n 5 6) compared tocontrols (n 5 5) was analyzed by hematoxylin-eosin, Nissl, and silver staining; relationshipsbetween regional vulnerability and oxidative metabolism were evaluated by cytochromeoxidase histochemistry. Profile counting-based estimates showed that 13% and 27% ofneurons in layers II/III and layers IV/V of somatosensory cortex had ischemic cytopathology,respectively; CA1 neuronal perikarya appeared undamaged, and ,10% of CA3 and CA4neurons were injured; and neuronal damage was 79% in putamen, 17% in caudate, butnucleus accumbens was undamaged. Injury was found preferentially in primary sensoryneocortices (particularly somatosensory cortex), basal ganglia (predominantly putamen,subthalamic nucleus, and substantia nigra reticulata), ventral thalamus, geniculate nuclei,and tectal nuclei. In sham piglets, vulnerable regions generally had higher cytochromeoxidase levels than less vulnerable areas. Postischemic alterations in cytochrome oxidasewere regional and laminar, with reductions (31–66%) occurring in vulnerable regions andincreases (20%) in less vulnerable areas. We conclude that neonatal hypoxia-ischemia causeshighly organized, system-preferential and topographic encephalopathy, targeting regions thatfunction in sensorimotor integration and movement control. This distribution of neonatalencephalopathy is dictated possibly by regional function, mitochondrial activity, and connec-tivity. J. Comp. Neurol. 377:262–285, 1997. r 1997 Wiley-Liss, Inc.

Indexing terms: neonatal encephalopathy; somatosensory system; basal ganglia; cytochrome

oxidase; O2 deprivation

Many newborn humans experience transient cerebralhypoxia-ischemia (H-I) during delivery or cardiorespira-tory arrest (Volpe, 1987). Encephalopathy and neurologicabnormalities, such as movement disorders (e.g., ataxia,choreoathetosis, diplegia, or dystonia), epilepsy and devel-opmental delay, are possible lifelong consequences thatoccur following perinatal H-I (Volpe, 1987). Preterm orterm H-I infants have damage in forebrain and brainstem(Schneider et al., 1975; Low et al., 1989; Rorke, 1992).Studies of H-I encephalopathy in newborn animal models

have implicated several mechanisms for neuronal degen-eration, including excitotoxicity, oxidative injury, and aci-dosis (Myers, 1977; Silverstein et al., 1986; Ikonomidou

Contract grant sponsor: U.S. Public Health Service; Contract grantnumbers: NS 20020, NS34100

*Correspondence to: Lee J. Martin, Ph.D., Johns Hopkins UniversitySchool of Medicine, Department of Pathology, Neuropathology Laboratory,720 Rutland Avenue, 558 Ross Research Building, Baltimore, MD 21205-2196. E-mail: lmartin@welchlink. welch. jhu.edu

Received 1 February 1996; Revised 22 July 1996;Accepted 6 September 1996

THE JOURNAL OF COMPARATIVE NEUROLOGY 377:262–285 (1997)

r 1997 WILEY-LISS, INC.

Fig. 1. Macrophotographs of representative Nissl-stained coronalsections from control (A–C) and H–I piglet brains with laminarnecrosis (D–F) or pan-laminar necrosis (G–I) in the neocortex. Similarpreparations were used to generate data in Figures 3–5. Arrows in Aidentify dorsal (d), ventral (v), medial (m), and lateral (l) orientations.Solid black arrows identify selective laminar (D–F) or pan-laminar

(G–I) necrosis in the somatosensory cortex. In G–I, note the medialand lateral expansion of damage in the neocortex. Open arrowheadsidentify damage in the putamen and caudate; note the medialexpansion of damage into the caudate in G–I. A, amygdala; C, caudate;GP, globus pallidus; M, medial septal nucleus; nbM, nucleus basalis ofMeynert; P, putamen; T, thalamus. Scale bar 5 2.1 mm.

BRAIN DAMAGE IN HYPOXIC-ISCHEMIC NEWBORNS 263

et al., 1989; Razdan et al., 1993). Although the neurologicaldisabilities of children that experienced transient cerebralO2 deprivation in early life suggest abnormalities incerebral cortex and/or basal ganglia, the vulnerabilities ofspecific neural systems and the pathophysiological mecha-nisms that dictate regional susceptibility to H-I in new-borns are still unknown.

Here, we used newborn pigs in a novel model of asphyxiccardiac arrest followed by cardiopulmonary resuscitationand return of spontaneous circulation to identify theregional distribution of brain damage caused by transientH-I. We specifically evaluated two hypotheses. First, be-cause children with cerebral palsy have prominent motorabnormalities (Volpe, 1987; Kuban and Leviton, 1994), wetested the hypothesis that the distribution of brain dam-age is regionally selective and is suggestive of a connectiv-ity-based, transneuronal vulnerability of neural systemsthat function in control of movement. Second, because highintrinsic regional cerebral blood flow and glucose consump-tion may correspond to the distribution of vulnerable brainregions in nonhuman primate models of neonatal asphyxia(Myers, 1972; Myers, 1977; Rorke, 1992), we determined,by using in situ enzyme histochemistry for cytochromeoxidase (CO), whether oxidative metabolism predicts re-gional vulnerability and whether regional changes inmitochondrial function occur postischemically. The resultsof this study demonstrate that this porcine model ofnewborn asphyxia is a suitable experimental animal modelto identify neuroanatomical, cellular and molecular mecha-nisms of perinatal encephalopathy caused by transientH-I. Such a model of cerebral O2 deprivation is relevantclinically because perinatal H-I has been estimated tooccur in 1–5% of all human births (Volpe, 1987).

MATERIALS AND METHODS

Animal model of pediatric H-I

The animal protocols used in this study were approvedby the Animal Care and Use Committee of the JohnsHopkins Medical Institutions. One-week-old piglets (,2.5kg) were anesthetized with sodium pentobarbital (65mg/kg, i.p.). The trachea was intubated and the lungs were

Fig. 2. H–I in piglets results in neuronal injury in the somatosen-sory cortex (A), striatum (B), and hippocampus (C) at 4 days recovery.Values are mean 6 standard deviation (SD) of estimates of thepercentage of neurons with ischemic cytopathology, as determined byprofile counting of incomplete samples of the different regions. Thesevalues do not reflect total neuronal numbers within the entire region.Asterisks indicate significant differences (P , 0.05) from controls.

Fig. 3. (facing page). Topographic distribution of brain injury foundin 2 out of 6 H–I piglets. A–F: Representative coronal sections from theforebrain (A is most anterior) through rostral midbrain (F is mostposterior). The midline is to the right. Solid black denotes areas ofnecrosis and hatching denotes areas of pre-necrosis (i.e., the presenceof ischemic neurons and inflammatory changes, but not elimination ofneurons as in necrosis). Laminar necrosis and pre-necrosis are local-ized to the parietal cortex (somatosensory neocortex). Prominentsubcortical damage is found in the putamen, ventral thalamus, andthe subthalamic nucleus. A, amygdala; AC, anterior commissure; C,caudate; GP, globus pallidus; H, hippocampus; IC, internal capsule;NA, nucleus accumbens; P, putamen; S, subthalamic nucleus; T,thalamus. Scale bar 5 2 mm.

264 L.J. MARTIN ET AL.

Figure 3

Figure 4

ventilated to maintain normal blood gases. Under sterileconditions, a femoral incision was made, and catheterswere advanced into the thoracic aorta and vena cava.Piglets received a maintenance infusion of 10 ml/kg/hrlactated Ringer’s solution. Incisions were closed and pig-lets received cephalothin. Fentanyl (10 µg/ml, i.v.) wasgiven during surgery to maintain anesthesia, and pancuro-nium bromide (0.3 mg/kg, i.v.) was administered to pro-duce muscle paralysis. Piglets were kept on a warmingblanket and covered with a plastic bag to maintain normalrectal temperature at 38.5–39.5°C. Baseline measure-ments of arterial blood gases, pH, hemoglobin content, O2content, glucose, lactate, and blood pressure were deter-mined. One group of piglets served as surgical shamcontrols (n 5 5). Other piglets (n 5 10) were exposed to 30minutes of hypoxia (arterial O2 saturation 30%), 5 minutesof room air (O2 saturation 65%) followed by 7 minutes ofairway occlusion (O2 saturation 5%). At 7 minutes ofasphyxia, ventilation was instituted with 100% O2, andepinephrine was injected as a bolus (0.1 mg/kg, i.v.)followed by continuous infusion (40 µg/kg/min). Sternalchest compressions were performed at a rate of 100/minwith a 50% duty cycle using a pneumatically driven piston(Thumper). Upon recovery of spontaneous circulation,sternal chest compressions were terminated, and whenarterial pressure exceeded 60 mmHg, epinephrine infu-sion was discontinued. Sodium bicarbonate was infused (1mEq/kg) to maintain arterial pH at 7.4. Fentanyl wasinfused (5 µg/kg/h) for 4 hours. Mechanical ventilation wasdiscontinued at 6 hours and animals were extubated.Blood gases were checked to assure adequate spontaneousventilation. Piglets generally awakened between 6–8 hourspostischemia and drank water between 12–24 hours. By24 hours, piglets usually drank formula milk. Animalswere kept in a 0.5 3 1 meter animal transport cage with awarming blanket and a bowl of water. They were observedat least twice during the first night and were taken out ofthe cage at least five times per day for bottle feeding. Ifanimals exhibited seizures (usually between 24–48 hours),they were treated with diazepam (5 mg).

Piglet brain preparation

Only the piglets that survived a 4-day recovery periodand were perfusion-fixed were used in this study. Thenumber of surviving animals in each group was as follows:nonischemic sham controls (n 5 5); and H-I piglets (n 5 6).Piglets were deeply anesthetized with sodium pentobarbi-

tal and then perfused (20 minutes) intraaortically withcold (4°C) 4% paraformaldehyde prepared in 0.1 M phos-phate buffer (pH 7.4) after clamping the descending aortaand exsanguination with phosphate-buffered saline. Afterfixation, the brains were bisected midsagittally, and eachhemisphere was cut into 1 cm slabs. From the left hemi-sphere, coronal samples were taken consistently at ante-rior striatal and mid-hippocampal levels. These sampleswere processed for paraffin histology. Neurodegenerationwas assessed in sections (10 µm) stained with hematoxylinand eosin (H&E) and silver impregnation (Yamamoto andHirano, 1986). The right hemisphere was cryoprotected inphosphate-buffered 20% glycerol for 24 hours, frozen inisopentane, and stored at 280°C. These samples were cutserially at 40 µm on a sliding microtome, and adjacentsections were used for cresyl violet (CV) staining, immuno-cytochemical localization of glial fibrillary acidic protein(GFAP), and enzyme histochemical assay of cytochromeoxidase (CO). Peroxidase anti-peroxidase methods wereused, as described previously (Martin et al., 1991), todetect GFAP with a monoclonal antibody (BoehringerMannheim, Indianapolis, IN) at a concentration of 40 ng/ml.

CO histochemistry

To identify the levels of oxidative metabolism on aregional and cellular basis, we used the CO histochemicalmethod of Wong-Riley (Wong-Riley, 1979). Brain sectionsfrom control and H-I piglets were prepared concomitantly.The enzymatic reaction medium was prepared freshly,consisting of 100 mM phosphate buffer (pH 7.4), 0.1%horse heart cytochrome C, 117 mM sucrose, and 1.4 mMdiaminobenzidine tetrahydrochloride. In this histochemi-cal reaction, in situ cytochrome oxidase catalyzes thetransfer of electrons (donated by diaminobenzidine) fromcytochrome C, provided as substrate, to O2 to form H2O.The donation of electrons from diaminobenzidine is achromogenic reaction yielding the formation of an in-soluble precipitate in the vicinity of CO activity (Wong-Riley, 1989). For negative controls, 10 mM KCN was addedto the reaction medium. Sections were incubated for 2.5hours at 37°C in a Dubnoff metabolic shaker incubator.After the reaction, sections were rinsed in phosphatebuffer, mounted on glass slides, and coverslipped.

Standards were used for densitometric quantification ofrelative CO activity in brain sections. The preparation ofstandards was similar to other methods (Gonzalez-Limaand Garrosa, 1991). Paraformaldehyde fixed brains werepulverized, acetone extracted, and air-dried. Brain powderwas resuspended in 100 mM phosphate buffer and boiled(15 minutes) to destroy endogenous CO activity. Knownunits of cytochrome oxidase (Sigma, St. Louis, MO) wereadded to equal amounts of brain paste (final concentra-tions ranged from 1 cu/mg to 200 u/mg) supplemented with0.1% glutaraldehyde for crosslinking to brain paste pro-teins. Agarose was added to the suspension to provide amatrix. Congealed standards were cryoprotected in buff-ered 20% glycerol, frozen in isopentane, and cut (40 µm) ona sliding microtome. Standards were always processedconcomitantly with piglet brain sections.

Data analysis

Profile counting was used to estimate ischemic neuronaldamage in H&E-stained paraffin sections. The regions

Fig. 4. A subgroup of H–I piglets (2 out of 6) deviated from thedistribution of damage observed in Figure 3 by having only minorneocortical laminar prenecrosis, but subcortical damage was virtuallyidentical to that shown in Figure 3. A–F: Representative coronalsections from the forebrain (A is most anterior) through rostralmidbrain (F is most posterior). The midline is to the right. Solid blackidentifies areas of necrosis and hatching identifies areas of pre-necrosis. Laminar pre-necrosis was found selectively in the parietalcortex (somatosensory neocortex). Prominent subcortical necrosis isfound in the putamen and the substantia nigra pars reticulata (SNR).Prominent subcortical pre-necrosis is found in the ventrolateralglobus pallidus, entopeduncular nucleus, ventral thalamus, and thesubthalamic nucleus. A, amygdala; AC, anterior commissure; C,caudate; EP, entopeduncular nucleus; GP, globus pallidus; H, hippocam-pus; IC, internal capsule; NA, nucleus accumbens; P, putamen; S,subthalamic nucleus; T, thalamus. Scale bar 5 2 mm.


Figure 5

analyzed were anterior, superior parietal cortex (somato-sensory cortex), septal hippocampus, central putamen,medial head of the caudate nucleus, and nucleus accum-bens (core). Profile counting, rather than stereology, wasused because we were unable to fulfill a systematic ran-dom sampling design at the inception of this study. Insections that were matched for level, the number ofneuronal profiles were counted in six non-overlapping,microscopic fields at 31,000. In each microscopic field, thefraction of neurons with ischemic cytopathology was deter-mined in each animal. Neurons were distinguished fromglial cells by morphological appearance. The criteria forischemic cytopathology included, eosinophilic cytoplasm,cytoplasmic vacuolation, and nuclear pyknosis. The sixvalues for each brain region in each animal were averaged,and this individual number was used to determine groupmeans. To chart the regional distribution of brain damagein H-I piglets, CV-stained sections were analyzed by usinga video-based mapping system (Martin et al., 1991). Sec-tion contours were outlined, and damaged areas wereidentified and plotted. Silver and immunocytochemicalpreparations were analyzed qualitatively for neurofibril-lary degeneration of neuronal cell bodies/terminals andastrocytic responses, respectively.

CO-stained brain sections were analyzed densitometri-cally by using an image-processing system (Loats Associ-ates, Westminster, MD) as described previously (Balloughet al., 1995). Slides were placed on a light box, and videoimages of sections (matched for level) were captured with aDage 68 video camera. Digitized images of piglet brainsections were then displayed in either gray scale orpseudocolor. For determination of optical density of reac-tion product, and thus relative CO activity, the system wascalibrated with the CO standards by measuring the graylevel of each standard. One optical density measurementfor each brain region was obtained from each piglet withincontrol and H-I groups. Constant-size field delimitingframes were used to measure the average-integrated opti-cal density over the majority of the region of interestwithin each section. With this approach, we have foundthat a single measurement over a relatively large area issimilar to multiple measurements within the same regionusing smaller field delimiting frames. Measurements ofthe same region in near-adjacent sections were similar.The linearity and interassay variability of the method was

estimated using five standards and three different histo-chemical experiments.

Statistical analysis of ischemic neuronal damage anddensitometric measurements were performed by usingone-way analysis of variance followed by a Student’s t-test.Probabilities of 5% were interpreted as statistically signifi-cant.

RESULTS

Gross neuropathology

Gross examinations of sham and H-I piglet brains wereunremarkable. The was no evidence of intracerebral hem-orrhage of any type. Cavitary and cystic lesions wereabsent at 4 days recovery. The gyral development was inaccord with the pattern expected at this age.

Brain injury in H-I newborns is systempreferential and organized topographically

In control piglets, the cytoarchitecture was normal inneocortex (Figs. 1A–C, 6A), hippocampus (Fig. 8A), basalganglia (Figs. 1A–C, 9A,B,H, 10A,C, 11A), and brainstem(Figs. 1C, 12A,C, 13A,C). This observation was based on asystematic, light microscopic analysis of serial 40-µm-thick sections (1 section in every 800 µm) stained with CVand paraffin sections stained with H&E and silver. Pri-mary sensory neocortical regions (i.e., somatosensory, audi-tory, and visual cortex) were characterized by a thick layerIV and a cell sparse zone in lower layer V (Fig. 6A). Thecaudate nucleus and putamen (Fig. 9A,B) had their charac-teristic cytology with few large neurons (30–40 µm indiameter) interspersed among the principal medium-sizedneurons (10–20 µm in diameter). Hippocampal (Fig. 8A)and thalamic (Figs. 12A,C, 13A,C) cytoarchitecture in con-trols was consistent with previous normative descriptionsof swine hippocampus (Holm and Geneser, 1991) andthalamus (Solnitzky, 1938). In H&E preparations of shambrains, background neuronal injury (possibly due to theanesthesia, perfusion-fixation, brain compression duringremoval from the skull, or histological processing) was,3% in neocortex, striatum, and hippocampus (Fig. 2).

Brain damage in H-I piglets was regionally selective andtopographic in distribution (Figs. 1D–I, 3–5). In CV-,H&E-, and silver-stained sections and in GFAP immunocy-tochemical preparations, injury was classified as eitherpre-necrotic or necrotic damage. Pre-necrotic damage (Figs.6F, 9D, 10B,D, 12B,G) was characterized by inflammatorychanges, including perivascular cuffing and perineuronalincrustations of leukocytes, with neurons showing cytoplas-mic eosinophilia (in H&E sections) or pallor (in CV sec-tions) and nuclear pyknosis. Early or pre-necrotic degenera-tion was also typified by neurofibrillary abnormalities(Fig. 7B) and loss of GFAP immunoreactivity (Figs. 7D,12H). In contrast, necrotic damage (Figs. 6B–E, 9C,E, 11B,13B,D) was characterized by elimination of neurons, spon-giform changes in the neuropil, terminal degeneration(Fig. 9G) and gliomesodermal reactive changes, includingastrogliosis with enhanced GFAP immunoreactivity (Fig.9F), proliferation of the microvasculature, and massiveparenchymal infiltration of inflammatory cells (neutro-phils, monocytes, or macrophages).

Subsets (13–27%) of neocortical neurons in H-I pigletshad ischemic cytopathology (Fig. 2A). In Nissl sections,

Fig. 5. Regional distribution of severe brain injury found in 2 out of6 H–I piglets. A–F: Representative coronal sections from the forebrain(A is most anterior) through rostral midbrain (F is most posterior). Themidline is to the right. Solid black identifies areas of necrosis andhatching identifies areas of pre-necrosis. Virtually complete necrosis isfound in the parietal cortex (somatosensory neocortex) and motorcortex, with damage extending medially into the cingulate cortex andlaterally to the inferior parietal cortex. Most of the putamen isseverely damaged with medialward expansion of damage into themajority of the caudate. The globus pallidus showed focal damage,whereas the entopeduncular nucleus was damaged more completely.Prominent subcortical damage is also found throughout the thalamus,subthalamic nucleus, and substantia nigra. A, amygdala; AC, anteriorcommissure; C, caudate; EP, entopeduncular nucleus; GP, globuspallidus; H, hippocampus; IC, internal capsule; NA, nucleus accum-bens; P, putamen; S, subthalamic nucleus; SNR, substantia nigra parsreticulata; T, thalamus. Scale bar 5 2 mm.


Fig. 6. Photomicrographs of Nissl-stained sections showing thecytoarchitecture of the primary sensory neocortex in control piglets (A)and neocortical damage in H–I piglets (B–F). Numbers (1–6) denotecortical layers. A: Control somatosensory cortex is characterized by arelatively thick layer 4 and a thin layer 5 that is divided into an upper,dense pyramidal cell zone and a deeper cell sparse zone. Scale bar 5100 µm (same for B and C). B: In 2 out of 6 H–I piglets, thesomatosensory cortex had selective laminar necrosis that spannedlayers 4 and upper 5 (arrows). C: Some piglets (2 out of 6) hadpan-laminar necrosis in the somatosensory cortex that spanned layers2–6 (arrow). Most neurons in these layers were eliminated and the

neuropil was enriched with inflammatory cells and perivascularleukocytes. D: In H–I piglets with severe neocortical damage, borderzones (solid black line) were identified between pan-laminar necrosis(left) and laminar necrosis of deep layers (right). Scale bar 5 75 µm. E:In layers 4 and 5 of somatosensory cortex of piglets with selectivelaminar necrosis, boundary zones (solid black line) between necrosis(left of line) and pre-necrosis (right of line) are divisible. These borderzones delineate the selective laminar localization of injury. Scale bar 560 µm. F: Pre-necrosis in upper layer 5 of H–I piglet neocortex. Theneuropil is enriched with inflammatory cells and perivascular leuko-cytes (arrowhead). Scale bar 5 60 µm.


Fig. 7. Early laminar pathology in the parietal cortex of H–Ipiglets is associated with neurofibrillary degeneration and astrocyticchanges. A,B: Near-adjacent, paraffin sections stained with H&E (A)and silver (B) show that necrosis in specific neocortical layers overlapswith neurofibrillary degeneration of pyramidal cells (arrowheads) and

terminal degeneration (arrow). Numbers (1–6) identify cortical layers.C,D: Near-adjacent, frozen sections stained for Nissl (C) or GFAP (D)show that early laminar pre-necrosis (arrow in C) is associated withastrocytic changes as evidenced by loss of GFAP immunoreactivity(arrow in D). Scale bar 5 78 µm in A (applies to B–D).


three patterns of neocortical pathology were observed. Onepattern, found in 4 out of 6 piglets, was characterized byselective laminar damage in superior parietal cortex (Figs.1D–F, 3, 4, 6B,E, 7). This laminar damage was localized inthe somatosensory cortex as identified by cytoarchitectonicand metabolic criteria (Figs. 6A, 14A–C, 15A) and wasmost prominent in layers IV and upper layer V (Fig. 6B).Cortical laminae exhibiting necrosis were contiguous toareas, designated as border zones (Fig. 6E), that displayedpre-necrotic stages of degeneration in layers IV and V.Border zones were, in turn, contiguous to cortical areasshowing no morphological change (Fig. 6E). With thesecond pattern, found in 2 out of 6 H-I piglets, laminar

damage in somatosensory cortex was not advanced tonecrosis, but pre-necrotic degeneration was present (Figs.4, 6F) with prominent loss of GFAP immunoreactivity inlayers undergoing incipient injury (Fig. 7C,D). The extentof laminar neocortical damage was usually 5–10 mmlateral to the midline and approximately 14 mm in theanterior-posterior axis to encompass most of the superiorparietal cortex. In 4 out of 6 piglets, occipital and superiortemporal cortices showed evidence of pre-necrosis in layerIV (not shown). However, frontal, insular, cingulate, infe-rior parietal, inferior temporal, and primary olfactorycortices were unremarkable. In contrast, the third pattern,found in 2 out of 6 H-I piglets, was typified by globalpan-laminar necrosis of neocortex (Figs. 1G–I, 5), encom-passing superior and inferior parietal cortex, parieto-occipital cortex, and medial frontal cortex (including motorcortex and cingulate cortex). Damage extended approxi-mately 15 mm lateral to the midline and .16 mm in theanterior-posterior axis. However, primary olfactory cortexand insular cortex remained unremarkable. Within somato-sensory cortex of these animals, all layers were damaged,with extensive neuronal injury in layers II, III, IV, V, andVI (Figs. 5, 6C). In regions of neocortex that borderednecrotic zones, layers I, II, and VI showed pre-necrosis,whereas layers III, IV, and V were selectively ablated (Fig.6D).

In 6 out of 6 H-I piglets, the basal ganglia were damaged(Figs. 1D–I, 3–5, 9–11). In piglets with selective laminardamage in somatosensory cortex, the central putamen wasinjured consistently, showing neuronal elimination (Fig.9C,E), terminal degeneration (Fig. 9G), and astrocytosis(Fig. 9F). In contrast, the lateral caudate showed pre-necrosis, and the medial caudate and nucleus accumbenswere unremarkable (Figs. 1D–F, 3, 4). In H&E sections,79% and 17% of neurons had ischemic cytopathology incentral putamen and caudate, respectively (Fig. 2B). Me-dium-sized striatal neurons were severely damaged,whereas a subset of large neurons was preserved (Fig. 9C).Basal ganglia regions that are known targets of medium-sized spiny striatal neurons, including the external globuspallidus (GPe), entopeduncular nucleus, substantia nigrapars reticulata (SNR), also showed focal pre-necrosis ornecrosis that was highly topographic and not random inlocation (Figs. 3C, 4C–E). For example, pre-necrosis wasfound in the ventrolateral GPe, and infarction was ob-served in the central SNR of H-I piglets (Figs. 4F, 11B). In

Fig. 8. Photomicrographs of the temporal hippocampus in Nissl-stained coronal sections of control (A) and H–I (B) piglets. CA1, CA4,and dentate gyrus (DG) are shown. CA1 layers are identified as: o,stratum oriens; p, stratum pyramidale; r, stratum radiatum; and lm,stratum lacuosum-moleculare. DG layers are identified as: m, molecu-lar layer; g, granule cell body layer; and i, infragranular layer. Thepyramidal and granule cell body layers are predominantly unchangedin H–I piglets (see Fig. 2C for % injury). However, the lacuosum-moleculare of CA1 and the molecular layer of dentate gyrus (corre-sponding to the distal dendrites of pyramidal and granule cells,respectively) and the infragranular layer (solid black arrowheads) inH–I piglets contain many small, non-neuronal cells, corresponding toactivated glial cells or infiltrated leukocytes. The hippocampal fissure(open arrow) contains blood vessels encrusted with leukocytes. Scalebar 5 400 µm.

Fig. 9. The striatum in H–I piglets is damaged severely. A,B: InNissl-stained sections, the cytoarchitecture of control putamen (A) andcaudate nucleus (B) consists principally of medium-sized neurons(open arrowheads) and fewer large neurons (solid black arrowheads).Scale bar 5 150 µm. C: In the H–I piglet putamen, the principalneurons (i.e., the medium-sized neurons) degenerate, whereas asubset of the large neurons is spared (solid black arrowheads). Theneuropil of the putamen exhibits infiltration of small inflammatorycells and perivascular incrustation of leukocytes (curved, open ar-rows). D: In the caudate of H–I piglets, medium-sized neurons aremostly present, but nodules of infiltrated inflammatory cells areobserved (solid curved arrows). E,F: In necrotic areas of the centralputamen, as identified by Nissl staining (E), prominent astrogliosisoccurred, as determined by glial fibrillary acidic protein (GFAP)immunoreactivity (F). Scale bar 5 70 µm. G,H: Silver staining showedterminal degeneration (arrowheads) in the putamen of H–I piglets (G)as compared to controls (H). Scale bar 5 40 µm.


Figure 9

Fig. 10. The subthalamic nucleus (STN) is damaged in H–Inewborn piglets. A,B: Nissl stained sections showing the STN incontrol (A) and H–I (B) piglets. Scale bar 5 350 µm. C,D: Nissl stainedsections showing extensive infiltration of small inflammatory cells(i.e., pre-necrosis) into the STN postischemia (D) compared to control

(C). Scale bar 5 87.5 µm. E,F: Cytochrome oxidase (CO) histochemis-try shows that, compared to control (E), oxidative metabolism isreduced in the STN postischemia (F) with loss of CO-positive neuronsand formation of axonal swellings (arrowheads). Scale bar 5 50 µm.


6 out of 6 H-I piglets, the subthalamic nucleus wasdamaged (Figs. 3F, 4E, 10B,D). In contrast, the medialglobus pallidus and substantia nigra pars compacta/ventral tegmental area appeared undamaged.

Focal damage to diencephalic and brainstem nucleioccurred in all H-I piglets (Figs. 3, 4, 12B,D,G,H, 13B,D).In thalamus, primary sensory relay nuclei were damagedin 6 out of 6 piglets. Somatosensory ventral tier nuclei (i.e.,ventral posterolateral and ventral posteromedial) weredamaged consistently, as evidenced by early pre-necroticdegeneration or necrosis (Fig. 12D,G,H). The pretectalnucleus, the medial geniculate nucleus (Fig. 13D), and thelateral geniculate nucleus (Fig. 13B) were consistentlypre-necrotic or infarcted at 4 days recovery. Motor compo-nents of thalamus (i.e., ventral anterior and ventral lateral

nuclei) displayed early inflammatory changes (Fig. 12B).In contrast, limbic, association, and nonspecific relaynuclei in anterior, midline, and posterior nuclear groups inthalamus were undamaged (Fig. 3). Hypothalamus wasunremarkable. In midbrain, the inferior and superiorcolliculi were necrotic, and the red nucleus showed changesconsistent with pre-necrosis (not shown). Periaqueductalgray, motor nuclei of cranial nerves, pons and inferior olivewere unremarkable.

In animals that showed global pan-laminar necrosis ofneocortex (2 out of 6 piglets), subcortical damage was moresevere than in piglets with only selective laminar damagein somatosensory cortex (Figs. 1G–I, 5). Basal forebrain,basal ganglia, diencephalic, and brainstem necrosis andgliomesodermal scarring were more extensive these pig-lets with global neocortical injury. These piglets containedinjured neurons and gliomesodermal scarring in the basalforebrain magnocellular complex (Fig. 1H), and showednecrosis of medial territories of caudate, central, anddorsomedial areas of globus pallidus, and complete infarc-tion of entopeduncular nucleus, SNR, and subthalamicnucleus. All primary sensory relay thalamic nuclei (i.e.,ventroposterior, lateral geniculate, and medial geniculatenuclei) and some limbic thalamic nuclei (midline nucleargroups and laterodorsal nucleus) were damaged (Fig. 5).

In contrast to the neocortex, basal ganglia, and primarysensory regions of brainstem in H-I piglets, limbic fore-brain regions were relatively spared at 4 days recovery.The lateral septum and bed nucleus-amygdala complexwere undamaged (Figs. 1, 3–5). The hippocampal forma-tion exhibited minimal changes. In H&E sections, neuro-nal cell bodies in CA1 appeared undamaged (Fig. 2C), and,10% of neurons in CA3 and CA4 had ischemic morpholo-gies (Fig. 2C). CV-stained sections confirmed that neuronswithin the pyramidal cell body layer in CA1 were pre-served in H-I piglets (Fig. 8B). Silver-stained sections ofhippocampus also showed no evidence for neurofibrillarydegeneration of CA1 neurons or terminal degeneration inH-I piglets (not shown). However, inflammatory changeswere present in the vicinity of the hippocampal fissure(Fig. 8B). Intraparenchymal monocytes and microgliawere enriched within the lacuosum-moleculare layer ofCA1 and infragranular layer of the dentate gyrus in H-Ipiglets (Fig. 8B).

Regionally selective metabolic defects andreorganization occur in the H-I newborn

brain

Enzyme histochemistry for in situ localization of COactivity was used to show that brain oxidative metabolismvaries on a regional basis in normal piglets and that H-Iproduces regional defects in oxidative metabolism in thenewborn brain following recovery. In control piglets, basalCO activity was not uniform throughout the brain, asevidenced by differential regional and laminar enrich-ments (Figs. 13E,F, 14A–C, 15A, 16A,C). Basal CO activitywas highest in primary sensory regions throughout theneuraxis in control piglets. For example, in neocortex, COactivity was higher in paracentral, superior temporal, andoccipital cortices than in limbic cortex (Figs. 14A–C,16A,C; Table 1). In somatosensory cortex, layer IV andupper layer V pyramidal neurons showed the greatestactivity for CO, whereas layer I exhibited the lowest CO

Fig. 11. The substantia nigra pars reticulata (SNR) is damaged inH–I newborns. Nissl stained sections show that, compared to control(A), the H–I piglet SNR (B) is infarcted as evidenced by the centralizednecrosis with extensive infiltration of inflammatory cells into theperiphery. Scale bar 5 344 µm.


Figure 12

activity (Fig. 15A). In thalamus, oxidative metabolism washigh in ventroposterior nuclei (Figs. 12E, 14C), lateralgeniculate nucleus (Fig. 13E), and medial geniculatenucleus (Fig. 13E). Intralaminar, limbic, and associationthalamic nuclei were less active than primary sensoryrelay nuclei, with the exception of the laterodorsal nucleus(Fig. 14C). In brainstem, superior and inferior colliculidisplayed high oxidative metabolism (Fig. 13F). Incuba-tion of brain sections with KCN (10 mM) included in thereaction medium completely inhibited the activity of CO(Fig. 15B).

Alterations in CO activity were present in H-I piglets(Figs. 10F, 12F, 14D–F, 15C,D, 16B,D; Table 1). Thedirection of change was region and intra-region specific.For example, the metabolic changes of the somatosensorycortex were differential within the gyral crown versus deepwithin the sulcus (Figs. 14D–F, 15C,D). In the gyral crownof primary somatosensory cortex (i.e., areas 1 & 2) inpiglets showing laminar injury, CO activity was reduced inlayers IV, V, and VI but increased in layers I, II, and III(Fig. 15C,D), whereas deep within the sulcus (i.e., areas3a,b), all layers showed marked declines in oxidativemetabolism. In contrast, all divisions of the somatosensorycortex of H-I piglets with pan-laminar necrosis showed asignificant reduction in all layers. Densitometric analysisof H-I piglet neocortex (average-integrated optical densityof all layers) revealed 31–61% reductions in CO activity inareas 1, 2 and areas 3a,b of somatosensory cortex (Table 1).Comparisons among adjacent CO- and Nissl-stained sec-tions demonstrated that loss of neocortical CO activity didnot always overlap with neuronal cell body injury, particu-larly in areas contiguous with somatosensory cortex (com-pare Figs. 1D–I and 14D–F). For example, CO activity wasreduced postischemia in superior temporal cortex (Fig.14F) without showing appreciable injury by Nissl (Fig.1F,I) and silver staining.

In contrast, mesocortical and allocortical limbic regionsof H-I piglets showed an elevation in oxidative metabolismas compared to controls. Cortical regions that showedenhanced staining for CO activity in the H-I brain in-cluded, primary olfactory cortex, cingulate cortex, gyrusrectus, and entorhinal cortex (Figs. 14D–E, 16B). How-ever, in some regions, these qualitative changes were notsubstantiated quantitatively by densitometry (Table 1). Inentorhinal cortex of H-I newborn pigs, neurons in layer IIand the neuropil of superficial layer I displayed a dramaticincrease in CO activity as compared to controls (Fig.16A,B). The postischemic metabolic activation of limbic

cortices was consistently greater in piglets with moresevere neocortical damage. The dentate gyrus molecularlayer (outer two-thirds and inner third), CA4, and CA3exhibited increases in metabolic activity postischemia(Fig. 16C,D). For example, CO activity in CA3 was in-creased (24%) in H-I piglets (Table 1). Histochemicallydetectable CO activity in CA1 was also increased (20%)postischemia (Table 1), primarily in the stratum lacuosum-moleculare and stratum oriens (Fig. 16C,D).

Subcortical regions also displayed differential alter-ations in oxidative metabolism at 4 days postischemia. COactivity in the putamen of H-I piglets was significantlyreduced (66%) relative to controls (Table 1). On a groupbasis, mean CO activity in caudate was unchanged (Table1), but on an individual basis, CO in caudate was moredepressed in H-I piglets with the greatest neocorticaldamage. However, rostral portions of caudate showedenhanced metabolism (Fig. 14D). Reduced CO activity wasalso observed in several thalamic nuclei (Figs. 12F, 14F)and in subthalamic nucleus (Fig. 10F). In ventral posteriorthalamic nuclei (Fig. 12F) and subthalamic nucleus (Fig.10F) CO-positive axonal swellings were observed fre-quently. In contrast, some subcortical limbic system struc-tures (e.g., nucleus accumbens and olfactory tubercle)were activated metabolically at 4 days postischemia (Fig.14D).

DISCUSSION

Transient cerebral H-I caused by perinatal asphyxia orneonatal cardiorespiratory arrest is a risk factor for chil-dren with cerebral palsy, developmental delay, and epi-lepsy (Volpe, 1987; Kuban and Leviton, 1994). Thesechildhood neurological disorders can be associated withmovement disorders, visual-motor learning abnormalities,and seizures (Volpe, 1987; Kuban and Leviton, 1994).Therefore, we tested the hypothesis that cerebral H-Iresults in system-selective damage in the newborn brain.We also evaluated relationships between regional vulner-ability and regional oxidative metabolism to identify aninherent property of specific brain systems that maycontribute to the selective vulnerability to H-I.

Brain damage in H-I newborns issystem-selective and organized

topographically

H-I in piglets preferentially damages primary sensoryand forebrain motor systems. The cerebral cortex andbasal ganglia are highly vulnerable. The neocortical areathat is most vulnerable to H-I corresponds to primarysomatosensory cortex based on electrophysiological map-ping of somatosensory-evoked potentials in newborn pigs(Woolsey and Fairman, 1959; Craner and Ray, 1991), andthe most vulnerable region of piglet striatum (i.e., centralputamen) is the sensorimotor-recipient region, based onknown corticostriatal connectivity in other mammals (Joneset al., 1977) and tracing studies in piglet (Martin andIchord, unpublished observations). In diencephalon, tha-lamic relay nuclei for somatosensory (ventral posteriornucleus), visual (lateral geniculate nucleus), auditory (me-dial geniculate nucleus), and motor (ventral anterior/lateral) systems are consistently damaged. In brainstem,visual (superior colliculus) and auditory (inferior collicu-

Fig. 12. Damage to ventral tier thalamic nuclei occurs followingneonatal H–I. A,B: By nissl staining, the ventral anterior thalamicnucleus in H–I piglets (B) shows parenchymal and perivascularinfiltration of small inflammatory cells compared to control (A). Scalebar 5 90 µm. C,D: In Nissl stained sections, the ventral posteriorlateral nucleus in H–I piglets (D) shows necrosis with gliomesodermalscarring compared to control (C). Scale bar 5 90 µm. E,F: COhistochemistry shows that, compared to control (E), oxidative metabo-lism is severely reduced in the ventral posterior lateral nucleuspostischemia (F) with low CO-activity in the neuropil and formation ofaxonal swellings (arrowheads). Scale bar 5 90 µm. G,H: Pre-necrosisof ventral thalamic nuclei (asterisk), as shown by Nissl staining (G),overlaps with zones in near-adjacent sections (H) showing reducedGFAP immunoreactivity (asterisk). Scale bar 5 100 µm.


Fig. 13. Comparison of the lateral geniculate (A,B) and medialgeniculate (C,D) nuclei of control (A,C) and H–I (B,D) newborn pigsshows that subcortical primary sensory relay nuclei are highly vulner-able to O2 deprivation. Neuronal degeneration and severe gliomesoder-mal scarring occur in both nuclei postischemia (arrows). Similardamage occurs in sensory relay nuclei in tectum (not shown). The

lateral and medial geniculate nuclei (E) and superior and inferiorcolliculi (F) in controls have high basal oxidative metabolism based onCO activity (dark areas). LGNd, lateral geniculate nucleus parsdorsalis; LGNv, lateral geniculate nucleus pars ventralis; MGN,medial geniculate nucleus; IC, inferior colliculus; SC, superior collicu-lus. Scale bars 5 100 µm (A,B); 150 µm (C,D); 500 µm (E,F).


lus) relay nuclei are predisposed to injury. This regionaldistribution neocortical and subcortical injury is impor-tant conceptually because it indicates that the formation ofH-I encephalopathy in newborns is not a random andstatic process but, rather, is highly organized and topo-graphic, targeting preferentially regions that function insensory-motor integration and control of movement. Thepattern of brain damage in H-I piglets bears a closeresemblance to that found in perinatal asphyxia in hu-mans, although the prominence of brainstem versus telen-cephalic injury varies (Schneider et al., 1972; Myers, 1977;Low et al., 1989; Rorke, 1992).

Our observations are similar to previous neuropathologi-cal studies of asphyxia neonatorum in experimental ani-mals, with notable exceptions. Other models of H-I innewborn pig show damage to forebrain (LeBlanc et al.,1991, 1995). Term or near-term monkeys subjected toepisodes (10–16 minutes) of total asphyxia and resuscita-tion exhibit damage primarily to diencephalic and brain-stem nuclei (Ranck and Windle, 1959; Myers, 1972, 1977).The inferior colliculus shows the greatest vulnerability,and other structures that function in primary sensoryrelay in medulla are vulnerable, including superior olives,trigeminal nerve sensory nuclei, dorsal column nuclei, andvestibular nuclei (Ranck and Windle, 1959; Myers, 1972,1977). In these nonhuman primate paradigms of totalasphyxia, forebrain damage was not observed frequently.This brainstem/thalamic distribution of injury is consis-tent with our results, but our model of H-I also exhibitsprominent forebrain damage. This difference betweenmodels may be related to the severity of O2 deprivation andthe presence or absence of postischemic convulsive sei-zures. Asphyxic cardiac arrest in newborn pigs is a hybridcombination of hypoxia and incomplete cerebral ischemiaand, thus, is a model of partial cerebral O2 deprivation. Inaddition, seizures occur commonly in H-I piglets. Interest-ingly, with monkey models, if animals exhibit convulsiveseizures or if near-term fetuses are subjected to episodes ofin utero partial asphyxia, the paracentral neocortex andbasal ganglia are damaged (Ranck and Windle, 1959;Myers, 1972, 1977), concordant with our results using amodel of asphyxic cardiac arrest in newborn pigs.

Interpretation of the system-selective braindamage in H-I newborns: The concept

of a transsynaptic organizer

The distribution of brain damage in H-I piglets isimportant because the pattern provides insight into mecha-nisms of injury. Vulnerable regions of the newborn telen-cephalon and brainstem are components of primary sen-sory and forebrain motor systems. These areas have highrates of blood flow and glucose consumption (Myers, 1972;Cavazutti and Duffy, 1982). Mitochondrial function is alsohigh in systems vulnerable to H-I in newborn pigs, consis-tent with previous studies of normal brain in other mam-mals (Arsenio-Nunes et al., 1973; Jones et al., 1986;Huntley and Jones, 1991; Hevner et al., 1995).

During cerebral H-I in newborns, rapid responses ofprimary sensory systems in brainstem may organize thedistribution of injury in forebrain sensorimotor systems.Depolarization of brainstem neurons (Haddad and Jiang,1993) and increased blood flow and glucose utilization inbrainstem sensory regions of newborns (Duffy et al., 1982;

Cavazzuti and Duffy, 1982) occur in response to hypoxia.Anoxic depolarization of neurons and glia occurs soon afterthe onset of H-I (Haddad and Jiang, 1993; Balestrino,1995), and release of glutamate can mediate neuronaldeath in anoxic neuronal cultures (Rothman, 1984). High-affinity glutamate uptake, a major function of astrocytes(Danbolt, 1994), is suppressed by perinatal H-I (Silver-stein et al., 1986), possibly causing transient elevations inextracellular glutamate in neocortex and striatum (Hag-berg et al., 1987; Gordon et al., 1991). Primary sensoryregions in brainstem are innervated by glutamatergicprimary afferents (Salt and Herrling, 1991), and theseregions have ascending glutamatergic connections viathalamus to layer IV of cerebral cortex (Salt and Herrling,1991). Corticostriatal (Jones et al., 1977) and corticosubtha-lamic (Fujimoto and Kita, 1993; Bevan et al., 1995)projections originating in layer V are also glutamatergic(Fonnum et al., 1981; Bevan et al., 1995) and terminatetopographically in striatum (Jones et al., 1977; Alexanderand Crutcher, 1990) and subthalamus (Bevan et al., 1995).However, ionotropic glutamate receptor subtypes and glu-tamate transporter subtypes have relatively similar local-izations within different regions of neocortex and differentareas of striatum (Martin et al., 1993a,b; Rothstein et al.,1994); yet, forebrain damage is predominantly found insomatosensory cortex and putamen in H-I piglets at 4 daysrecovery. The finding that pre-necrosis is associated withloss of the astroglial marker GFAP and astroglial gluta-mate transporter (Martin et al., 1996) supports the involve-ment of astrocytic abnormalities in the onset of neurodegen-eration in H-I newborns. Thus, regional localization offunction, mitochondrial activity, and connectivity are pos-sible organizers of the localization of brain damage innewborns following H-I. Adaptive physiological responsesin brainstem and astroglial dysfunction may have adverseeffects by transiently maintaining excitatory synaptic func-tion selectively in primary sensory pathways, therebycausing a topographic cascade of excitotoxic, transneuro-nal encephalopathy preferentially in systems involved insensorimotor integration.

The piglet CA1 tolerates H-I, contrasting with theselective vulnerability of CA1 to ischemia in adult experi-mental animals and humans (Kirino, 1982; Petito et al.,1987). Other studies of perinatal H-I in newborn dogs(Mujsce et al., 1993) and rats (Schwartz et al., 1992) alsoshow tolerance in CA1. This observation supports ourregional metabolism-connectivity hypothesis for the orga-nization of brain injury in H-I newborns. For example, COactivity is lower in piglet CA1 than in highly vulnerableregions. Therefore, the lower intrinsic oxidative metabo-lism of the immature CA1 may be protective during H-I.These neurons may also be different intrinsically fromadult CA1 neurons with respect to afferent connectivityand postsynaptic signal transduction mechanisms, or theymay require more time to degenerate. Alternatively, angio-architecture may explain the selective distribution of H-Ibrain damage (Graham, 1992).

Forebrain metabolism is alteredin newborns following H-I

Postischemic reductions in oxidative metabolism werefound in relation to different neuropathological patterns.Depletion of CO activity was observed in necrotic and


Figure 14

pre-necrotic regions, reflecting injury and elimination ofneurons and afferents as supported by H&E, Nissl, andsilver staining. Decreased CO in regions that were appar-ently undamaged based on H&E and Nissl staining sug-gests that mitochondrial defects in neurons or astrogliamay be early abnormalities. Alternatively, these regionsmay have reduced CO due to loss of presynaptic terminalsresulting from denervation; however, this explanation isnot supported by silver staining of terminal degeneration,which occurs after or concurrently with detectable damageto neuronal cell bodies.

Metabolic activation of entorhinal-hippocampal circuitsand other limbic/autonomic regions occurred followingH-I. This finding may reflect synaptic reorganization dueto damage in primary sensory neocortical and forebrainmotor regions (Kaas et al., 1983; Kolb and Whishaw, 1989)or activation due to status epilepticus (Tanaka et al., 1992;White and Price, 1993). Alternatively, postischemic hyper-metabolism in entorhinal-hippocampal circuits mayforeshadow brain injury. Ischemic neuronal damage inadult CA1 is preceded by enhanced glutamate receptor-mediated synaptic excitation (Urban et al., 1989) andhypermetabolism (Jorgensen et al., 1990). Because theserial glutamatergic connectivity between layer II of ento-rhinal cortex, dentate gyrus/CA3, and CA1 is established(Rosene and Van Hoesen, 1987), elevated CO in entorhinalcortex and hippocampus is consistent with transsynapticactivation (Wong-Riley, 1989). Mild neuronal injury in CA3and CA4 and inflammatory changes in the lacuosum-moleculare of CA1 and molecular layer of dentate gyrus(corresponding to the distal dendrites of pyramidal andgranule cells, respectively) occurred in H-I piglets. Thesechanges may reflect excitotoxic somatodendritic injury,because swelling and degeneration of distal dendrites areinitial responses of excitotoxic neuronal cell death (Olneyet al., 1972).

Seizures may contribute to the distributionof brain damage in H-I newborns

Most H-I piglets had overt seizures 24–48 hours postisch-emia. Prolonged (2 hours) seizures in neonatal pig cancause progressive hypotension, decreased cardiac output,and reduced left ventricular contractility (Young et al.,1985), suggesting interactions among incomplete cerebralischemia, cardiac dysfunction and status epilepticus. TheSNR frequently underwent necrosis in H-I piglets. Dam-age to this region is consistent with status epilepticus butnot H-I (Nevander et al., 1985; Auer and Siesjo, 1988). Incontrast, striatal damage is characteristic of H-I but notstatus epilepticus (Nevander et al., 1985; Auer and Siesjo,1988). The globus pallidus is usually damaged completelyby seizure activity (Nevander et al., 1985), althoughdamage to this region was subtotal in H-I piglets. Thecontribution of H-I or status epilepticus to the localizationof damage in somatosensory cortex and ventral posteriorthalamus is less clear. Similar to the distribution ofdamage in these regions in piglets, status epilepticus inwell-oxygenated adult rats causes selective neuronal in-jury in layer IV of somatosensory cortex and in ventral tiernuclei of thalamus, but, unlike our observations in H-Ipiglets, other primary sensory regions in epileptic rats arenot affected regularly (Nevander et al., 1985). Further-more, focal sensorimotor seizures cause lesions primarilyin layer IV of somatosensory cortex and in thalamic nucleithat have reciprocal connections with the epileptogenicfocus in cortex (Collins and Olney, 1982). Although thesubset of piglets that seized were subsequently treatedwith diazepam, the temporal relationship between theonset of clinical seizures and the formation of brain injuryis uncertain. In a rodent model of seizures, diazepampretreatment is highly effective in abolishing seizure-related brain lesions (Martin et al., 1985). Administrationof diazepam to H-I piglets after the onset of seizure activityis probably less effective than prophylactic administrationin ameliorating seizure-related injury. Differences in therelative severity of status epilepticus, as well as thepresence or absence of cardiovascular complications (Younget al., 1985), may cause variations in the extent of braindamage in H-I newborns.

CONCLUSIONS

This pediatric model of asphyxic cardiac arrest causesan encephalopathy consistent with combined H-I andseizure-related insults. Primary sensory and basal gangliasystems are damaged preferentially. Regional connectivity

TABLE 1. Regional CO Alterations in the H-I Newborn Brain at 4 DaysRecovery

Region Control H-I

Somatosensory cortex (areas 1 and 2) 148.0 6 21.61 102.7 6 15.3*Somatosensory cortex (area 3a, b) 126.0 6 8.2 49.3 6 11.5*Cingulate cortex 76.0 6 33.7 79.3 6 20.8Putamen 106.0 6 14.1 36.0 6 23.5*Caudate 81.0 6 7.1 74.0 6 23.9CA1 81.0 6 7.1 101.0 6 7.0*CA3 66.0 6 10.1 86.0 6 8.2

1Values (in units of CO enzyme/mg brain tissue) are mean 6 standard deviation basedon one average-integrated optical density measurement from each region in each animal(see Materials and Methods).*Values are significantly different (P , 0.05) from sham-controls.

Fig. 14. CO enzyme histochemistry reveals the differential re-gional metabolic activity in control piglet brain (A–C) and the region-ally differential metabolic deficits and activation in H–I piglet brain(D–F). Arrows in A identify the dorsal (d), ventral (v), medial (m), andlateral (l) orientation of these coronal sections. The intensity ofblack/white reflects the level of CO activity, with the most blackindicating the highest level of metabolism and the most whiteindicating the lowest level of metabolism. See Table 1 for densitom-etry. A–C: In control piglets, basal metabolism is highest in theparietal cortex (solid black arrowheads), corresponding to the primarysomatosensory cortex, and moderate in other neocortical regions.Basal metabolism is moderate to high in the putamen, caudate,septum and the ventral thalamus and lowest in the hypothalamus.D–F: Postischemia, regional oxidative metabolism is reduced mostmarkedly in somatosensory cortex (solid black arrowheads), butreductions also occur in other neocortical regions (e.g., the inferiorparietal and superior temporal cortices). CO activity is depressed inthe putamen, caudate and the ventral thalamus. In contrast, somelimbic/autonomic structures show increased metabolism relative tocontrols. Postischemic hypermetabolism occurs in the primary olfac-tory cortex (small, curved arrowheads), olfactory tubercle, gyrusrectus, nucleus accumbens, and the entorhinal cortex (open arrow-head). A, amygdala; Acb, nucleus accumbens; C, caudate; OT, olfactorytubercle; P, putamen; S, septum; H, hypothalamus; VPL, ventroposte-rior thalamic nucleus. Scale bar 5 1.6 mm.


Fig. 15. CO enzyme histochemistry shows the basal metabolicactivity in control piglet somatosensory cortex (A) and the laminar/intraregional metabolic changes in H–I piglet somatosensory cortex (Cand D). Numbers (1–6) denote cortical layers. The intensity ofblack/white reflects the level of CO activity, with the most blackindicating the highest level of metabolism, and the most white indicatingthe lowest level of metabolism (e.g., subcortical white matter). See Table 1for densitometry. A: In control somatosensory cortex, the highest level ofoxidative phosphorylation is in layer 4, while the lowest level is inlayer 1. Some pyramidal neuron cell bodies (arrowheads) in upper

layer 5 show high metabolic activity. B: Negative control section showsthat preincubation with KCN inhibits uniformly all detectable COactivity (open arrowhead identifies the pial surface). C: In the crown(corresponding to areas 1 and 2) of somatosensory cortex in H–Ipiglets, CO activity is selectively reduced in deeper layers (4–6), whilesupragranular layers are hypermetabolic relative to control. Somemetabolically-active pyramidal neuron cell bodies are present (arrow-head). D: Deep within the sulcus of the somatosensory cortex (corre-sponding to areas 3a and b) of H–I piglets, metabolism is dramaticallyreduced in the neuropil and cell bodies of all layers. Scale bar 5 87 µm.


and mitochondrial function are possible determinants ofthe system-preferential vulnerability, and the concept of atranssynaptic organizer of H-I encephalopathy in thenewborn was introduced. We hypothesize that H-I innewborns causes a topographic cascade of excitotoxicity-mediated transneuronal injury in brain systems involvedin sensorimotor integration. Connectivity studies in H-Ipiglets are necessary to validate this hypothesis. Thesimilarities in the distribution of brain damage in H-Ipiglets compared to human newborns indicate that thisporcine model of pediatric cardiac arrest will be useful foridentifying mechanisms of perinatal H-I brain injury andthe neurologic consequences. The prominent and highlyreproducible postischemic damage to basal ganglia (e.g.,putamen, SNR, and subthalamic nucleus) is particularlyexciting because these regions function in control of move-

ment. Long-term survival experiments are necessary toestablish whether this localization of brain damage in H-Inewborn pigs causes persistent neurologic abnormalitiesthat are reminiscent of cerebral palsy (e.g., ataxia, choreo-athetosis, diplegia, or dystonia) and epilepsy.

ACKNOWLEDGMENTS

The authors thank Ann Price and Dawn Spicer forsuperb technical assistance. This work was supported byU.S. Public Health Service grants (NS 20020, NS 34100).

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Fig. 16. Transynaptic metabolic activation of entorhinal cortical-hippocampal pathways in H–I piglets is suggested by the localizationof CO activity. A,B: In the entorhinal cortex (numbers identify layers),the neuropil of the superficial part of layer 1 and neurons in layer 2(arrows) become hypermetabolic postischemia (B) relative to control(A). Scale bar 5 200 µm. C,D: In the hippocampus, the molecular (open

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primary sensory and forebrain motor systems in the newborn brain are preferentially damaged by...

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