Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 7-Day Trial for You or Your Team.

Learn More →

Selective Vulnerability of Subplate Neurons after Early Neonatal Hypoxia-Ischemia

Selective Vulnerability of Subplate Neurons after Early Neonatal Hypoxia-Ischemia 3308 • The Journal of Neuroscience, April 15, 2003 • 23(8):3308 –3315 Selective Vulnerability of Subplate Neurons after Early Neonatal Hypoxia-Ischemia 1,2 1 2 3 Patrick S. McQuillen, R. Ann Sheldon, Carla J. Shatz, and Donna M. Ferriero 1 2 3 Departments of Pediatrics and Neurology, University of California San Francisco Medical Center, San Francisco, California 94143-0106, and Department of Neurobiology, Harvard Medical School, Boston, Massachusetts 02115 Neonatal hypoxia-ischemia in the preterm human leads to selective injury to the subcortical developing white matter, which results in periventricular leukomalacia (PVL), a condition associated with abnormal neurodevelopment. Maturation-dependent vulnerability of late oligodendrocyte progenitors is thought to account for the cellular basis of this condition. A high frequency of cognitive and sensory deficits with decreasing gestational age suggests pervasive abnormalities of cortical development. In a neonatal rat model of hypoxic- ischemic injury that produces the characteristic pattern of subcortical injury associated with human PVL, selective subplate neuron death is seen. The premature subplate neuron death occurs after thalamic axons have reached their targets in cortex. Thus, as expected, thalamocortical connections form normally, including patterned connections to somatosensory cortex. However, deficits in motor function still occur, as in babies with PVL. Subplate neuron cell death in PVL provides another mechanism for abnormal neurodevelop- ment after neonatal hypoxia-ischemia. Key words: periventricular leukomalacia; visual; cortex; development; premature infant; thalamocortical creasing gestational age (Piecuch et al., 1997). Cortical visual im- Introduction pairment (i.e., visual loss caused by impairment of posterior vi- Hypoxia-ischemia (H-I) results in selective damage to different sual pathways) is particularly common, especially in infants with brain structures depending on the developmental stage at which PVL, in whom estimates range from 66% (Lanzi et al., 1998) to it occurs (Johnston, 1998). H-I in the preterm human [gestation- 94% in infants with severe PVL (Cioni et al., 1997). al week (GW) 23–32] causes damage to subcortical developing Subplate neurons are required for normal visual cortical de- white matter, a condition known as periventricular leukomalacia velopment (for review, see Allendoerfer and Shatz, 1994). They (PVL) (Volpe, 2001b). Developmental immaturity of the cerebral are among the first generated cells of neocortex (Luskin and vasculature is thought to account for this characteristic subcorti- Shatz, 1985) and come to lie beneath the developing cortical cal distribution (Volpe, 2001a). Other mechanisms include selec- plate, where they participate in the earliest neocortical circuitry tive cellular vulnerability, which relates to intrinsic properties of (Friauf and Shatz, 1991). Subplate neurons undergo pro- the vulnerable cell type. Cells of the oligodendrocyte lineage grammed cell death and are primarily absent from adult neocor- manifest stage-specific vulnerability to H-I (Back et al., 2002) tex (Chun et al., 1987). In humans, the subplate zone peaks in size through mechanisms of oxidative stress (Oka et al., 1993; Back et at GW 24, when the subplate is four times the width of the devel- al., 1998) and excitotoxicity (Matute et al., 1997; Fern and Moller, oping cortical plate (Kostovic and Rakic, 1990), and declines 2000; Follett et al., 2000). Subplate neurons, a transient cell type thereafter. The peak of subplate development coincides with the located beneath the cortical plate (Chun et al., 1987), are also gestational age associated with the highest incidence of PVL. vulnerable to stage-specific excitotoxicity (Chun and Shatz, Given their critical role in normal visual cortical development, 1988). the death of subplate neurons after H-I would illuminate mech- H-I in the preterm infant disrupts normal development and anisms that account for the high incidence of cortical visual im- results in significant cerebral injury. Neurological disability is pairment observed in PVL and provide a general model for ab- observed in 51% of premature infants (GW 25 weeks) exam- normal cortical development after H-I. In the present study, we ined at 30 months of age (Wood et al., 2000) and persists into examine a neonatal rodent model of H-I brain injury (Sheldon et adulthood (Hack et al., 2002). Deficits are found in motor, per- al., 1998) to determine whether subplate neurons die after neo- ceptual, and cognitive systems (Volpe, 2001b). These widespread natal H-I. abnormalities of cerebral development can be measured quanti- tatively with advanced magnetic resonance imaging (Miller et al., 2002). Cognitive impairment is associated significantly with de- Materials and Methods Animals. Timed-pregnant Sprague Dawley rats (Simonsen, Gilroy, CA) were allowed food and water ad libitum. All animal research was ap- Received Oct. 24, 2002; revised Jan. 24, 2003; accepted Jan. 29, 2003. proved by the University of California San Francisco Committee on An- This research was supported by National Eye Institute Grants EY02858 (C.J.S.) and NS35902 (D.M.F.) and National imal Research and was performed in accordance with standards of hu- Institutes of Health Grant K08 HD01396-03 (P.S.M.). We thank Cynthia Cowdrey for preparing cryostat sections and mane care set forth in the Policy on Humane Care and Use of Laboratory Michael DeFreitas and Gabriel Zada for assistance with bromodeoxyuridine, in situ end labeling, and double staining. Animals. Correspondence should be addressed to Dr. P. S. McQuillen, Department of Pediatrics, Box 0106, University of Hypoxia-ischemia. The manipulation was performed at postnatal day 1 California San Francisco Medical Center, San Francisco, CA 94143-0106. E-mail: [email protected]. Copyright © 2003 Society for Neuroscience 0270-6474/03/233308-08$15.00/0 (P1) or P2 (day of birth  P0) as described previously (Sheldon et al., McQuillen et al. • Subplate Neurotoxicity after Hypoxia-Ischemia J. Neurosci., April 15, 2003 • 23(8):3308 –3315 • 3309 1996). The first three litters received H-I at P1, and mortality was 31% ously (Parent et al., 1999). Primary antibody dilutions used were 1:200 (10 of 32). These animals were used for in situ end labeling (ISEL) and for anti-GFAP (G-A-5; Sigma) and 1:1000 for BrdU (mouse monoclonal; bromodeoxyuridine (BrdU) staining (Figs. 1–3). Because mortality was Roche Molecular Biochemicals). Primary antibody was applied over- lower (19%), and the pattern of injury by ISEL staining was unchanged, night at room temperature. Secondary antibody (biotinylated donkey subsequent litters received H-I at P2. These animals were used for anal- anti-mouse; Jackson ImmunoResearch) was applied for 1 hr at room ysis of thalamocortical innervation and motor testing (Figs. 4, 5). Total temperature. The sections were washed and developed with a mouse Elite mortality was 25% (16 of 64). To produce ischemia, pups were anesthe- ABC kit (Vector Laboratories, Burlingame, CA). tized with nitrous oxide, halothane, and oxygen. A midline incision was BrdU counting. The subplate zone was identified as described previ- made in the neck; the right common carotid artery was dissected from the ously (McQuillen et al., 2002) by accepted criteria (Boulder Committee, jugular vein and permanently coagulated with a bipolar coagulator 1970) and cytoarchitectonic features of neocortex (Bayer and Altman, [common carotid ligation (CCL)]. Sham-operated animals received the 1990). Specifically, in the radial domain, the subplate (layer VIB) was same operation, except that the carotid artery was not coagulated. Ani- localized at the base of the cortical plate, immediately below layer VI mals were returned to the dam for 1–2 hr. Subsequently, pups receiving neurons, and contained characteristic pyramidal neurons. The borders hypoxia were placed in 5.6% oxygen in chambers floating in a 37°C water in the coronal plane were determined by the characteristic six-layered bath for 3 hr. One pup from each litter was monitored with a skin surface neocortex and extended to cingulate cortex in the medial direction and temperature probe to ensure consistency between litters and that the entorhinal cortex laterally. Only heavily BrdU-labeled cells that fell into animals did not overheat. The body surface temperature was kept con- this region were counted. As in previous studies (Price et al., 1997), stant at 34°C (normal P7 core temperature, 35–37°C). Six litters of ro- heavily labeled cells were defined as cells in which more than half the dents, representing 64 animals, were used for the study. Rats were killed nucleus was labeled. Littermates were analyzed for quantification of sub- with pentobarbital, 100 mg/kg, given by intraperitoneal injection. plate neuron death. Brains from animals receiving H-I at P1 (n  6), Subplate neuron BrdU birth dating. Timed-pregnant Sprague Dawley sham-operated animals (n 3), or unmanipulated animals (n 2) were rats received an intraperitoneal injection of BrdU, 30 mg/kg (Sigma, St. analyzed at P5. Despite the use of timed-pregnant rats, significant vari- Louis, MO), at embryonic day 12.5 (E12.5; plug date, E0.5) to label ability occurs between litters in BrdU uptake and labeling of subplate subplate neurons in neocortex (Bayer and Altman, 1990). neurons. Therefore, no attempt was made to compare absolute numbers Fluorescent BrdU staining. BrdU-labeled subplate neurons were visu- of BrdU-positive neurons among experimental groups. Cell counts were alized as described previously (McQuillen et al., 2002). Brains were re- expressed as the interhemispheric ratio of BrdU-labeled subplate neu- moved rapidly from the cranium and flash-frozen in Tissue-Tek O.C.T. rons (BrdU-positive cells ipsilateral to CCL/BrdU-positive cells con- compound (Sakura Finetek, Torrance, CA) in a dry ice–95% ethanol tralateral to CCL). Cell death is expressed as a percentage derived from bath. Coronal cryostat sections (10 m thick) were fixed in 0.1 M sodium this ratio (100  ipsilateral/contralateral  100). The brains were sec- phosphate-buffered 4% paraformaldehyde, extracted with 0.6% Triton tioned entirely, and a random series representing every 10th section was X-100, acetylated, quenched in 3% hydrogen peroxide, and dehydrated selected and analyzed. There were no differences between groups in the through graded alcohols. To expose incorporated BrdU, the sections number of sections per animal (85  8.4 vs 82  8.4 sections per brain, were microwaved for 10 min in 0.1 M sodium citrate, pH 5.0. Anti-BrdU mean  SD; p  0.57); therefore, average cell death per section is re- antibody (IU4; Caltag, Burlingame, CA) was applied at 1:20,000 with ported. The subplate was identified as described above in digital images, exonuclease III (ExoIII) 100 U/l (Roche Molecular Biochemicals, Indi- and heavily labeled BrdU-positive cells were counted in each hemisphere anapolis, IN) in ExoIII buffer supplemented to 100 mM NaCl with 1% in each section with the cell-counting macro (ftp://rsbweb.nih.gov/pub/ bovine serum albumin at 37°C for 1 hr. After washing, horseradish nih-image/user-macros/). peroxidase-conjugated goat anti-mouse secondary antibody (Jackson Perl’s iron stain. Free-floating sections were stained in a 1:1 mixture of ImmunoResearch, West Grove, PA) was applied at 1:200 in blocking 2% v/v HCl and 2% potassium ferrocyanide for 30 min at room temper- solution (supplied by the manufacturer) for 30 min, followed by tyra- ature (Connor et al., 1995). The sections were rinsed in distilled water mide signal amplification (TSA) (Direct-green; PerkinElmer Life Sci- twice for 10 min, and then precipitated iron was visualized with DAB, 0.5 ences, Boston, MA). The sections were counterstained with 0.001% mg/ml, in 0.1 M phosphate buffer with 0.07% hydrogen peroxide for 5–10 bis-benzimide. min. The sections were rinsed in 0.1 M PBS and mounted on slides, ISEL staining. To visualize dying cells, we used a modified version of dehydrated, and put under cover glass. the ISEL method (Blaschke et al., 1996), performed as described pre- Cytochrome oxidase staining. Free-floating sections were stained for viously (McQuillen et al., 2002). A reaction mixture containing 1 M cytochrome oxidase activity (Anderson et al., 1975) in a reaction mixture biotin-deoxyuridine 5-triphosphate (Roche Molecular Biochemicals), that contained 5 mg of DAB, 10 mg of cytochrome C (Sigma), 750 mg of 0.15 U/ml terminal transferase (Invitrogen, Rockville, MD), 1 terminal sucrose, 20 mg of catalase (Sigma), and 9 ml of 0.05 M sodium phosphate transferase buffer, and 1% bovine serum albumin was applied, and the buffer, pH 7.4. Sections were incubated overnight at 37°C, rinsed in 0.1 M sections were incubated for 1 hr at 37°C. Sections were washed and then PBS, mounted on slides, dehydrated, and put under cover glass. incubated with NeutraLite avidin– horseradish peroxidase (Molecular Carbocyanine dye labeling. Rats were fixed by transcardial perfusion Probes, Eugene, OR) at 1:1000 in blocking solution (supplied with the with 0.1 M sodium phosphate-buffered 4% paraformaldehyde or TSA Direct kit) for 30 min. The sections were washed and developed with immersion-fixed in the same fixative (embryonic ages). Brains were re- TSA Direct-Cy3 (PerkinElmer Life Sciences). Double labeling was per- moved and stored in fixative with 0.02% sodium azide. Small (100 formed with ISEL reacted before microwave treatment and BrdU pri- m), similar-sized crystals of DiI (D-282; Molecular Probes) or 1,1- mary antibody incubation. Visualization with direct TSA was performed dioctadecyl-3,3,3,3-tetramethylindodicarbocyanine perchlorate (DiD; sequentially, with inactivation of peroxidase between development of D-307; Molecular Probes) were placed into visual cortex. Alternate con- ISEL and BrdU immunohistochemistry. Sections of neonatal rat thymus figurations of dyes were placed in each hemisphere to distinguish ipsilat- (positive) and liver (negative) were analyzed as controls for the sensitivity eral from contralateral hemispheres. The dye was allowed to transport at of ISEL staining. 37°C for 3 weeks. Coronal sections were cut at 50 –100 monavi- DAB immunohistochemistry. Animals were perfused transcardially bratome. Sections were counterstained with 0.001% bis-benzimide. with 0.1 M phosphate buffer followed by cold 4% paraformaldehyde/0.1 Imaging. Digital images were acquired with a Nikon (Mellville, NY) M phosphate buffer. Perfused brains were cryoprotected in 25% sucrose Eclipse 800 microscope and a cooled CCD camera (Spot2; Diagnostic in 0.1 M phosphate buffer before being sectioned on a sliding microtome. Instruments, Sterling Heights, MI). Digital images were analyzed on an Fifty micrometer sections were quenched with 3% hydrogen peroxide, Apple (Cupertino, CA) G4 computer with the public domain NIH Image washed, and blocked (5% horse serum, 5% fish skin gelatin, and 0.1% program (developed at the National Institutes of Health and available on Triton X-100 in 0.1 M Tris-buffered saline). For BrdU staining, DNA was the Internet at http://rsb.info.nih.gov/nih-image/). denatured by incubating sections in 2N HCl at 37°C for 30 min and then Confocal montage imaging. Fluorescent cryosections (BrdU/ISEL) washed in 0.1 M sodium borate, pH 8.5, for 10 min as described previ- were imaged on a Leica (Cambridge, UK) confocal microscope. Low- 3310 • J. Neurosci., April 15, 2003 • 23(8):3308 –3315 McQuillen et al. • Subplate Neurotoxicity after Hypoxia-Ischemia magnification montages were reconstructed from 5 or 10 fields with Leica Qwin montaging software and a motorized stage. Complete sec- tions represent a montage of 8 –10 separate fields. Motor testing. Animals were raised to 3 months of age and then as- sessed for motor deficits (Crawley, 2000). Each animal was observed for gross deficits with open-field locomotion. Then animals were scored on a three-point scale (2, normal; 1, impaired; and 0, incapable of performing task) for rod and beam walking (1-inch-diameter rod, 1-inch-wide beam) and for the number of foot faults on stair climbing. Each animal was tested three times on each test, and the median value was analyzed for significance. Finally, gait analysis was performed with footprint pattern. Animals receiving H-I were compared with controls consisting of both unmanipulated littermates and sham-operated animals. Statistics. Normally distributed data are reported as mean  SD. Hy- pothesis testing for differences between groups was performed with an unpaired, two-tailed t test. Nonparametric data and percents are re- ported as mean  interquartile range (subplate cell death) or mean of median scores (motor testing) SEM. Differences between groups were determined with Mann–Whitney U test and corrected for ties. Results P1 rodent H-I results in subcortical, periventricular cell death To determine the pattern of cell death after CCL and hypoxia at P1 (H-I), we used fluorescent ISEL for detection of DNA strand breaks associated with dying cells, combined with a nuclear coun- terstain to visualize anatomy. After H-I, cells may die with mor- phologic features of necrosis or apoptosis (Northington et al., 2001). We use ISEL staining solely as a sensitive indicator of cell death, with the knowledge that cells dying with either phenotype may display an ISEL signal (Charriaut-Marlangue and Ben-Ari, 1995). DNA fragmentation can be detected normally in the brains of postnatal rats (Spreafico et al., 1995) and represents naturally occurring cell death during development. This normal pattern of cell death can be appreciated in sham-operated and unmanipulated littermates (data not shown) in the same pattern observed in the hemisphere contralateral to the carotid ligation in this model (Fig. 1 A–C, right hemispheres). As has been noted previously (Vannucci, 1990), hypoxia or ischemia alone does not result in any detectable increase in cell death. Superimposed on this naturally occurring cell death in the hemisphere ipsilateral to CCL is cell death that resulted from H-I (Fig. 1 A–C, left hemi- spheres). At 12 hr after manipulation, cell death caused by H-I peaked, and a broad band of cell death could be appreciated in the subcortical regions that contained subplate, intermediate, and subventricular zones (Fig. 1 B,C). The ventricular zone was af- fected but to a lesser degree. A smaller amount of cell death was also apparent scattered throughout the neocortex, and retrosple- nial cortex was especially affected (Fig. 1 B, asterisk). Within the thalamus, the reticular nucleus and internal capsule showed in- creased cell death (Fig. 1 B, arrowheads). In the hippocampus, dying cells were not observed in the pyramidal layer in any hip- pocampal subdivision. Scattered dying cells were noted in the hippocampal subgranular layer in both hemispheres. This pat- tern of cell death was present throughout the rostral-caudal ex- tent of cerebral cortex, beginning at the anterior commissure and extending into occipital cortex (Fig. 1 A–C). Cell death in the intermediate zone was most intense in parietal cortex at the level Figure 1. Subcortical topography of cell death 12 hr after P1 H-I. Dying cells with DNA strand of thalamus but extended to more caudal brain regions, including breaks are detected with ISEL 12 hr after H-I at P1. A–C, ISEL signal (red) and bis-benzimide visual and visual association areas, following the dorsolateral as- nuclear counterstain (blue) are shown alone in coronal sections from frontal ( A), parietal ( B), pect of the lateral ventricles (Fig. 1 A–C). Temporally, ISEL attrib- and occipital ( C) regions. Arrowheads mark cell death in reticular thalamus. Asterisk marks cell utable to H-I could be detected as early as the end of the hypoxic death in retrosplenial cortex. period in animals that died during the procedure (data not shown). By 12 hr after hypoxia, the ISEL signal peaked (Fig. 1); it diminished by 24 hr (Fig. 2), and by 4 d after H-I, increased cell McQuillen et al. • Subplate Neurotoxicity after Hypoxia-Ischemia J. Neurosci., April 15, 2003 • 23(8):3308 –3315 • 3311 (compare Fig. 2, compare C, D). There was a corresponding increase in ISEL- labeled dying cells in the subplate and in- termediate zone ipsilateral to CCL (Fig. 2 A,C), although the amount of cell death was less than at 12 hr after manipulation (Fig. 1). Using BrdU immunohistochem- istry in combination with ISEL labeling, we identified double-labeled cells that indi- cated dying subplate neurons (Fig. 2 B,C, vertical arrows). These cells were noted fre- quently only in the hemispheres receiving H-I. In animals receiving H-I in which dou- ble labeling was performed (n  4), we found significantly more double-labeled dy- ing subplate neurons ipsilateral to CCL than contralateral (4.5 1.9 vs 0.4 0.5 cells per section; p 0.0001). Quantification of subplate neuron cell death after H-I There is significant interanimal variability in the amount of damage after neonatal H-I (Rice et al., 1981; Sheldon et al., 1996, 1998; Towfighi et al., 1997). To quantify the extent and variability of subplate neu- Figure 2. Cell death and BrdU staining for subplate neurons 24 hr after P1 H-I. ISEL (red), BrdU immunohistochemistry (green), ron cell death after H-I, we examined and bis-benzimide nuclear counterstain (blue) in coronal sections taken from parietal cortex 24 hr after H-I at P1. A, Low- BrdU-positive cell density 4 d after H-I at magnification views showing both hemispheres. Large white boxes in A delineate higher-magnification views in D of contralateral hemisphere (hypoxia) and in C of ipsilateral hemisphere (H-I), and small white box indicates region shown in B that demonstrated P1 using systematic random sampling and single ISEL-positive (horizontal arrow), BrdU-positive (asterisk), and double-positive (vertical arrow) cells. SP, Subplate. digital imaging to quantify subplate neu- ron cell death per section (see Materials and Methods). Subplate neuron cell death death was not detectable (data not shown). Cell death was not was increased significantly by H-I (45  48 vs 2  9%, mean assayed at time points beyond 96 hr. interquartile range; p  0.006, Mann–Whitney U test; Fig. 3A). To confirm this pattern of subcortical injury with other methods, The severity of injury in any given animal was readily apparent we performed GFAP immunohistochemistry and Perl’s iron stain- from inspection of either the BrdU labeling or cresyl violet stain- ing 4 d after H-I at P1 (data not shown). Both GFAP immunohisto- ing and ranged from mildly increased death (Fig. 3D) to nearly chemistry and iron staining were increased in the subplate and in- complete death of heavily BrdU-labeled subplate neurons (Fig. termediate zones in exactly the distribution of the heaviest ISEL 3B). The variable neuronal injury that resulted from this manip- staining. Cresyl violet staining of neocortex was remarkably normal ulation, with injury to subplate neurons even in the mildest cases, despite the presence of scattered ISEL, and the histologic appearance confirms the selective vulnerability of subplate neurons relative of cortex did not reveal prominent cell loss (Fig. 3B–D). Animals to other neuronal populations at this age. with severe injury did show some loss of lower-layer neurons in neocortex (Fig. 3B). Thalamocortical connections form normally after neonatal H-I Subplate neurons die after neonatal H-I BrdU labeling suggests that significant numbers of subplate neu- To determine whether subplate neurons specifically were among rons die prematurely after neonatal H-I. To determine the effects dying cells, we permanently labeled subplate neurons as they of this premature subplate neuron cell death on thalamocortical were generating from dividing neuroblasts with a labeled nucle- development, it was first necessary to determine whether otide, BrdU (BrdU birth dating). Subplate neurons are generated thalamocortical connections formed normally. Given the signif- from dividing neuroblasts from E10.5 to E12.5 (Bayer and Alt- icant cell death in the intermediate and subventricular zones, it is man, 1990). A pulse of BrdU is taken up by dividing neuroblasts possible that there was significant injury to the developing in S-phase. Only neurons generated from the next round of cell thalamocortical and corticofugal axons. Excitotoxic ablation of division (i.e., subplate and marginal zone neurons) receive heavy subplate neurons in cat, before ingrowth of thalamic axons into BrdU label. The BrdU is diluted with each successive round of cell visual cortex, prevents normal innervation of their targets in layer division, so that later-generated neurons (e.g., layer VI-II) receive IV (Ghosh et al., 1990). However, ablation immediately after progressively lighter label. Immunohistochemistry can then de- innervation does not result in the absence of thalamic innerva- tect BrdU in heavily labeled subplate neurons at any age. BrdU tion of layer IV (Ghosh and Shatz, 1992). In rat, thalamic axons birth dating is currently the optimal method for identifying sub- have reached neocortex by P0 and innervate appropriate targets plate neurons (Allendoerfer and Shatz, 1994). over the subsequent days (Catalano et al., 1996). On the basis of At 24 hr after H-I, a comparison of BrdU staining in the hemi- these observations, we predicted that H-I at P2 would not disrupt sphere ipsilateral to CCL with the contralateral hemisphere visual thalamocortical innervation. Indeed, this is what we ob- showed a significant decrease in BrdU-labeled subplate neurons served using the lipophilic carbocyanine dyes DiI and DiD to 3312 • J. Neurosci., April 15, 2003 • 23(8):3308 –3315 McQuillen et al. • Subplate Neurotoxicity after Hypoxia-Ischemia trace thalamocortical connections at P7 after H-I at P2. At these ages, label in thal- amus resulting from dye placement in cortex is a combination of retrograde la- beling of thalamic neurons and antero- grade labeling of the descending cortico- thalamic projections. After dye crystal placement into visual cortex (n 5), there was robust label in the lateral geniculate nucleus (visual thalamus) in both hemi- spheres (Fig. 4C,D). Furthermore, despite extensive cell death observed in the inter- nal capsule (Fig. 1 B), labeled fibers could be observed coursing normally through the intermediate zone and turning medi- ally into the internal capsule before enter- ing thalamus (Fig. 4 A,B). Ablation of subplate neurons immedi- ately after innervation of layer IV prevents the activity-dependent refinement of thalamocortical connections into ocular dominance columns (Ghosh and Shatz, 1992). Rodent visual cortex contains monocular and binocular zones but no finer organization of ocular dominance (Antonini et al., 1999). To assess the de- velopment of patterned thalamocortical Figure 3. Quantification of BrdU-positive subplate neurons 4 d after H-I at P1. A, Plot of subplate neuron cell death (see connections after H-I at P2, we visualized Materials and Methods) comparing animals receiving H-I with controls (sham-operated and unmanipulated littermate; p the topographical whisker barrel repre- 0.006). Severely ( B), moderately ( C), and mildly ( D) affected examples of subplate neuron BrdU immunohistochemistry and cresyl violet staining from sections used for quantification of subplate neuron death are shown. Plotted percent cell death for each sentation in somatosensory cortex at P10. example (B, C, D) is indicated by letter in A. SP, Subplate. The whisker barrel representation is con- solidated over the first postnatal week we observed faults of the left rear paw most frequently in animals (O’Leary et al., 1994), and genetic manipulations of glutamater- receiving right CCL and hypoxia. These observations indicate that gic (Cases et al., 1996) and serotonergic (Vitalis et al., 1998) neu- despite normal-appearing open-field locomotion, P2 H-I leads to rotransmission disrupt barrel formation. In every case we exam- significant motor deficits. The relationship of these motor deficits to ined at P10 (n  4), after H-I at P2, well formed cytochrome subplate cell death remains to be elucidated. oxidase patches appeared in layer IV of somatosensory neocortex ipsilateral to CCL (Fig. 4 E) that were identical to those observed in Discussion the contralateral hemisphere (Fig. 4 F). We conclude from these analyses that P2 H-I does not disrupt These results demonstrate that a rat model of early neonatal H-I initial thalamocortical pathfinding and innervation of somato- leads to significant, premature subplate neuron cell death. De- sensory and visual cortex. Moreover, the initial development of spite intense subcortical injury to the developing subplate and patterned connections in somatosensory cortex proceeds nor- intermediate zones, thalamocortical connections to somatosen- mally. Our analysis does not address subsequent refinement and sory and visual cortex form normally. This manipulation results plasticity of sensory thalamocortical connections. in measurable motor deficits in mature animals. These data, combined with observations of injury to oligodendrocyte pro- P2 H-I results in motor deficits genitors (Back et al., 2002), support H-I at P2 in rat as a model of Human PVL is characterized by dysmyelination and spastic di- human PVL. This model is useful for testing the hypothesis that plegia, a static motor deficit with more pronounced involvement neonatal H-I disrupts neurodevelopment through effects on cor- of lower extremities than of upper extremities (Volpe, 2001b). To tical plasticity and the refinement of cortical connections as a determine whether H-I at P2 leads to motor deficits, we allowed result of subplate neuron cell death. animals from two litters to develop to maturity after H-I at P2. Animals were then observed during open-field locomotion and Maturation-dependent topography of H-I cerebral injury gait analysis, but they did not display any observable deficits (data H-I brain injury produces age-dependent and region-specific in- not shown). However, when tested on rod and beam walking, jury. Many factors contribute to the evolution of this pattern, animals receiving H-I performed significantly worse than controls including maturation of cerebral oxygen and substrate delivery (Fig. 5; rod walking score, 1.3 0.2vs0.7 0.1, control vs H-I, mean through development of the cerebral vasculature and autoregu- of median scores  SEM; tied p  0.04, Mann–Whitney U test; lation (Volpe, 2001a), selective cellular vulnerability related to beam walking score, 1.8  0.1 vs 1.3  0.1; tied p  0.02). To oxidative metabolism (Ferriero, 2001), excitatory amino acid sig- confirm this observation, rats were tested for foot faults while they naling (Johnston et al., 2001; Jensen, 2002), and programmed cell climbed an inclined stair. H-I-treated animals had more faults than death (Han et al., 2000; Northington et al., 2001). The Levine controls (Fig. 5; 2.4 0.3 vs 0.2 0.1; p 0.0001, Mann–Whitney model [i.e., right CCL followed by hypoxia (Levine, 1960)] per- U test). Although faults were not recorded by extremity, qualitatively formed on immature rats (Rice et al., 1981) at selected ages (Shel- McQuillen et al. • Subplate Neurotoxicity after Hypoxia-Ischemia J. Neurosci., April 15, 2003 • 23(8):3308 –3315 • 3313 Figure 4. Thalamocortical projection to somatosensory and visual cortex after P2 H-I. A–D, Thalamocortical and corticothalamic axons labeled with DiI and DiD crystals placed in auditory or visual cortex. Coronal sections at the level of internal capsule (A, B) or lateral geniculate nucleus (C, D) demonstrate labeled fibers and cell bodies. To distinguish ipsilateral/H-I hemi- sphere from contralateral/hypoxic hemisphere, the dyes were placed in opposite configuration. For this example, the ipsilateral hemisphere (A, C) had DiI (red) placed in visual cortex and DiD (green) placed in auditory cortex. The contralateral hemisphere (B, D) had DiD (green) placed in visual cortex and DiI (red) placed in auditory cortex. Axons can be seen traversing the internal capsule (A, B), and neurons in lateral geniculate (visual thalamus) are back-labeled (C, D)inan identical manner in the two hemispheres. E, F, Cytochrome oxidase staining of sensory thalamo- cortical axons in patchy representation of whisker barrels in hemisphere receiving H-I ( E) and hypoxia alone ( F). don et al., 1996; Towfighi et al., 1997) also produces age- dependent and region-specific brain injury. Analyzing H-I at P1 with ISEL, we clearly show a subcortical pattern of cell death Figure 5. Motor deficits and abnormal myelination in mature animals after H-I at P2. Animals similar to human PVL. In agreement with others (Towfighi et al., receiving H-I (n 11) performed significantly worse on motor testing (rod walking, tied p 0.04; 1997), we did not see significant hippocampal injury at this age, a beam walking, tied p 0.02; and stair climbing, p 0.0001) than controls [unmanipulated litter- distinct difference from H-I at P7 and later, when hippocampal mate (n 8) and sham-operated (n 2)]. For rod and beam walking, score 2, normal; 1, impaired; region CA3 becomes vulnerable to injury before the adult pattern and 0, unable to perform task. For stair climbing, animals were scored for the number of foot slips of CA1 injury occurs beginning at P21. With this sensitive assay while climbing. Values are mean of the median score SEM. HI, Hypoxia-ischemia. for cell death, we did note low levels of diffuse cell death in neo- cortex and retrosplenial cortex. In the most severely affected an- imals, lower cortical layers begin to show laminar cell loss. In even ternatively, this could represent altered cortical development af- the mildest cases, subplate neuron cell death occurred, which ter injury to subcortical areas. confirms the selective vulnerability of these neurons relative to other neuronal populations. We did not note columnar patterns Selective cellular vulnerability: subplate neurons of cell death that are frequently observed at P7 or laminar injury Many authors have speculated that changing patterns of brain to cortical layers III and V as occurs at P13 (Towfighi et al., 1997). injury during development relate to intrinsic properties of the Most descriptions of the pathology of PVL indicate that neocor- affected cell type, i.e., selective cellular vulnerability (Mattson et tex is spared (Banker and Larroche, 1962). However, using sen- al., 1989; Johnston, 1998; Volpe, 2001a). Late oligodendrocyte sitive quantitative volumetric techniques with magnetic reso- progenitors are the prototypical examples of such a cell type, nance imaging of human infants with PVL, several studies (Inder because they manifest stage-specific vulnerability in vitro to exci- et al., 1999; Peterson et al., 2000) noted marked reduction in totoxicity (Matute et al., 1997; Fern and Moller, 2000), oxidative cerebral cortical gray matter volume throughout the brain, which stress (Oka et al., 1993; Back et al., 1998), and oxygen– glucose raises the possibility that neuronal cell death in neocortex may be deprivation (Fern and Moller, 2000) and in vivo to H-I (Back et more widespread than has been appreciated in human PVL. Al- al., 2002). In humans, preoligodendrocytes are the predominant 3314 • J. Neurosci., April 15, 2003 • 23(8):3308 –3315 McQuillen et al. • Subplate Neurotoxicity after Hypoxia-Ischemia cell type in the oligodendrocyte lineage during the developmental ruption of column formation after subplate neuron ablation is not period of peak incidence of PVL (Back et al., 2001). known, one possibility was suggested by the effects of subplate neu- Subplate neurons share many of these same properties. Sub- ron ablation on BDNF expression (Lein et al., 1999). Kainate injec- plate neurons are a transient cell population (Chun et al., 1987; tion leads to a long-lasting increase in BNDF expression localized to Al-Ghoul and Miller, 1989; Price et al., 1997), and they undergo the region of subplate ablation. Increased BDNF is associated with programmed cell death in the first postnatal week in mice (Mc- alterations in the phenotype of cortical inhibitory neurons, leading Quillen et al., 2002). Subplate neurons are located beneath the to the suggestion that subplate neurons modulate activity- developing neocortex (Luskin and Shatz, 1985; Kostovic et al., dependent competition by regulating levels of neurotrophins and 2002) near areas of diffuse white matter signal abnormality seen excitability in the developing cortex. on magnetic resonance imaging in preterm human infants Abnormal or delayed myelination is the hallmark of PVL. H-I at (Maalouf et al., 2001) at risk for the diffuse type of PVL (Volpe, P7 depletes the subventricular zone of oligodendrocyte progenitors 2001b). In humans, the subplate zone peaks at the onset of the (Levison et al., 2001). However, H-I at P2 and P7 results in the developmental window of vulnerability to PVL (GW 24) and proliferation of reactive late oligodendrocyte progenitors (Back et undergoes dissolution during the third trimester, and subplate al., 2002), and decreased myelin basic protein expression is only neurons are largely absent after 6 months of postnatal age (Kos- transient after H-I at P7 (Liu et al., 2002). Although cortical visual tovic and Rakic, 1990). Subplate neurons undergo programmed impairment is associated frequently with abnormalities on magnetic cell death to a much greater extent than other cortical neurons resonance imaging, 71% of premature infants with moderate PVL (Price et al., 1997). Subplate neurons are vulnerable to excito- during the neonatal period were found to have at least one abnor- toxic cell death (Chun and Shatz, 1988), which allows for the mality of visual testing at 1 year of age, and yet 66% of these children selective ablation of subplate neurons after injection of the gluta- had normal optic radiations, and all had normal-appearing visual mate agonist kainate into embryonic and postnatal kittens cortex (Cioni et al., 1997). These findings underscore the need to (Ghosh et al., 1990; Ghosh and Shatz, 1992). At later time points, examine fully the neurobiology of neonatal H-I and its impact on cortical neurons become sensitive to kainate, and the injections cortical development. Subplate neuron injury, alone or coincident no longer result in a selective ablation of subplate neurons with oligodendrocyte injury, could explain these observations. (Ghosh and Shatz, 1994). Subplate neurons become incorpo- References rated into mature synaptic networks, receiving excitatory input Al-Ghoul WM, Miller MW (1989) Transient expression of Alz-50 immuno- from thalamus and making excitatory connections with neurons reactivity in developing rat neocortex: a marker for naturally occurring in layer IV of neocortex, as well as sending recurrent collateral neuronal death? Brain Res 481:361–367. projections back to thalamus (Friauf and Shatz, 1991). In the Allendoerfer KL, Shatz CJ (1994) The subplate, a transient neocortical present study, we have demonstrated that P2 H-I leads to mod- structure: its role in the development of connections between thalamus erate to near-complete subplate neuron cell death, whereas most and cortex. Annu Rev Neurosci 17:185–218. Anderson WA, Bara G, Seligman AM (1975) The ultrastructural localization of cortical neurons are left intact. The mechanism of this selective cytochrome oxidase via cytochrome. J Histochem Cytochem 23:13–20. vulnerability of subplate neurons to H-I is unknown but may Antonini A, Fagiolini M, Stryker MP (1999) Anatomical correlates of func- relate to early cellular maturation (Chun and Shatz, 1989), with a tional plasticity in mouse visual cortex. J Neurosci 19:4388 – 4406. developmentally related increase in glutamate receptor expres- Back SA, Gan X, Li Y, Rosenberg PA, Volpe JJ (1998) Maturation- sion, including NMDA receptor 1 (Catalano et al., 1997) and dependent vulnerability of oligodendrocytes to oxidative stress-induced AMPA and kainate receptors (Furuta and Martin, 1999). Nota- death caused by glutathione depletion. J Neurosci 18:6241– 6253. bly, most of these observations have been made in vivo. We have Back SA, Luo NL, Borenstein NS, Levine JM, Volpe JJ, Kinney HC (2001) Late oligodendrocyte progenitors coincide with the developmental win- described recently a method for purifying subplate neurons that dow of vulnerability for human perinatal white matter injury. J Neurosci will be useful in elucidating cellular mechanisms of subplate neu- 21:1302–1312. ron cell death in vitro (DeFreitas et al., 2001). Back SA, Han BH, Luo NL, Chricton CA, Xanthoudakis S, Tam J, Arvin KL, Holtzman DM (2002) Selective vulnerability of late oligodendrocyte Abnormal cortical development after neonatal H-I: role of progenitors to hypoxia-ischemia. J Neurosci 22:455– 463. subplate neuron cell death Banker BQ, Larroche JC (1962) Periventricular leukomalacia of infancy. Arch Neurol 7:386 – 410. The involvement of subplate neurons in neonatal H-I brain in- Bayer SA, Altman J (1990) Development of layer I and the subplate in the rat jury is significant in light of the role that subplate neurons play in neocortex. Exp Neurol 107:48 – 62. normal cortical development (for review, see Allendoerfer and Blaschke AJ, Staley K, Chun J (1996) Widespread programmed cell death in Shatz, 1994). Human thalamocortical development begins at the proliferative and postmitotic regions of the fetal cerebral cortex. Devel- time of the development of layers in the lateral geniculate nucleus opment 122:1165–1174. at GW 22–25 (Hitchcock and Hickey, 1980). Synaptogenesis in Boulder Committee (1970) Embryonic vertebrate central nervous system: revised terminology. Anat Rec 166:257–261. human visual cortex occurs between GW 28 and birth (Hutten- Cases O, Vitalis T, Seif I, De Maeyer E, Sotelo C, Gaspar P (1996) Lack of barrels locher et al., 1982). In rodent, somatosensory thalamic afferents in the somatosensory cortex of monoamine oxidase A-deficient mice: role of have reached cortex at P0 (Catalano et al., 1996) and innervate a serotonin excess during the critical period. Neuron 16:297–307. their targets in layer IV soon thereafter. Thus, our model and Catalano SM, Robertson RT, Killackey HP (1996) Individual axon mor- human PVL occur at a stage of visual cortical development most phology and thalamocortical topography in developing rat somatosen- analogous to the early postnatal cat when thalamic afferents have sory cortex. J Comp Neurol 367:36 –53. entered visual cortex but have not segregated into ocular domi- Catalano SM, Chang CK, Shatz CJ (1997) Activity-dependent regulation of NMDAR1 immunoreactivity in the developing visual cortex. J Neurosci nance columns. Ablation at this point in development disrupts 17:8376 – 8390. the activity-dependent refinement of thalamocortical connections Charriaut-Marlangue C, Ben-Ari Y (1995) A cautionary note on the use of into mature ocular dominance columns (Ghosh and Shatz, 1992). the TUNEL stain to determine apoptosis. NeuroReport 7:61– 64. Ocular dominance columns form through an activity-dependent Chun JJ, Shatz CJ (1988) A fibronectin-like molecule is present in the devel- competition (for review, see Goodman and Shatz, 1993), possibly for oping cat cerebral cortex and is correlated with subplate neurons. J Cell neurotrophins. Although the cellular and molecular basis of the dis- Biol 106:857– 872. McQuillen et al. • Subplate Neurotoxicity after Hypoxia-Ischemia J. Neurosci., April 15, 2003 • 23(8):3308 –3315 • 3315 Chun JJ, Shatz CJ (1989) The earliest-generated neurons of the cat cerebral Levine S (1960) Anoxic-ischemic encephalopathy in rats. Am J Pathol cortex: characterization by MAP2 and neurotransmitter immunohisto- 36:139 –147. chemistry during fetal life. J Neurosci 9:1648 –1667. Levison SW, Rothstein RP, Romanko MJ, Snyder MJ, Meyers RL, Vannucci SJ (2001) Hypoxia/ischemia depletes the rat perinatal subventricular zone Chun JJ, Nakamura MJ, Shatz CJ (1987) Transient cells of the developing mammalian telencephalon are peptide-immunoreactive neurons. Nature of oligodendrocyte progenitors and neural stem cells. Dev Neurosci 23:234 –247. 325:617– 620. Cioni G, Fazzi B, Coluccini M, Bartalena L, Boldrini A, van Hof-van Duin J Liu Y, Silverstein FS, Skoff R, Barks JD (2002) Hypoxic-ischemic oligoden- droglial injury in neonatal rat brain. Pediatr Res 51:25–33. (1997) Cerebral visual impairment in preterm infants with periventricu- Luskin MB, Shatz CJ (1985) Studies of the earliest generated cells of the cat’s lar leukomalacia. Pediatr Neurol 17:331–338. visual cortex: cogeneration of subplate and marginal zones. J Neurosci Connor JR, Pavlick G, Karli D, Menzies SL, Palmer C (1995) A histochem- 5:1062–1075. ical study of iron-positive cells in the developing rat brain. J Comp Neurol Maalouf EF, Duggan PJ, Counsell SJ, Rutherford MA, Cowan F, Azzopardi D, 355:111–123. Edwards AD (2001) Comparison of findings on cranial ultrasound and Crawley JN (2000) What’s wrong with my mouse? Behavioral phenotyping magnetic resonance imaging in preterm infants. Pediatrics 107:719 –727. of transgenic and knockout mice. New York: Wiley-Liss. Mattson MP, Guthrie PB, Kater SB (1989) Intrinsic factors in the selective DeFreitas MF, McQuillen PS, Shatz CJ (2001) A novel p75NTR signaling vulnerability of hippocampal pyramidal neurons. Prog Clin Biol Res pathway promotes survival, not death, of immunopurified neocortical 317:333–351. subplate neurons. J Neurosci 21:5121–5129. Matute C, Sanchez-Gomez MV, Martinez-Millan L, Miledi R (1997) Gluta- Fern R, Moller T (2000) Rapid ischemic cell death in immature oligoden- mate receptor-mediated toxicity in optic nerve oligodendrocytes. Proc drocytes: a fatal glutamate release feedback loop. J Neurosci 20:34 – 42. Natl Acad Sci USA 94:8830 – 8835. Ferriero DM (2001) Oxidant mechanisms in neonatal hypoxia-ischemia. McQuillen PS, DeFreitas MF, Zada G, Shatz CJ (2002) A novel role for Dev Neurosci 23:198 –202. p75NTR in subplate growth cone complexity and visual thalamocortical Follett PL, Rosenberg PA, Volpe JJ, Jensen FE (2000) NBQX attenuates ex- innervation. J Neurosci 22:3580 –3593. citotoxic injury in developing white matter. J Neurosci 20:9235–9241. Miller SP, Vigneron DB, Henry RG, Bohland MA, Ceppi-Cozzio C, Hoffman Friauf E, Shatz CJ (1991) Changing patterns of synaptic input to subplate C, Newton N, Partridge JC, Ferriero DM, Barkovich AJ (2002) Serial and cortical plate during development of visual cortex. J Neurophysiol quantitative diffusion tensor MRI of the premature brain: development in 66:2059 –2071. newborns with and without injury. J Magn Reson Imaging 16:621– 632. Furuta A, Martin LJ (1999) Laminar segregation of the cortical plate during Northington FJ, Ferriero DM, Graham EM, Traystman RJ, Martin LJ (2001) corticogenesis is accompanied by changes in glutamate receptor expres- Early neurodegeneration after hypoxia-ischemia in neonatal rat is necro- sion. J Neurobiol 39:67– 80. sis while delayed neuronal death is apoptosis. Neurobiol Dis 8:207–219. Ghosh A, Shatz CJ (1992) Involvement of subplate neurons in the forma- O’Leary DD, Ruff NL, Dyck RH (1994) Development, critical period plas- tion of ocular dominance columns. Science 255:1441–1443. ticity, and adult reorganizations of mammalian somatosensory systems. Ghosh A, Shatz CJ (1994) Segregation of geniculocortical afferents during Curr Opin Neurobiol 4:535–544. the critical period: a role for subplate neurons. J Neurosci 14:3862–3880. Oka A, Belliveau MJ, Rosenberg PA, Volpe JJ (1993) Vulnerability of oligo- Ghosh A, Antonini A, McConnell SK, Shatz CJ (1990) Requirement for sub- dendroglia to glutamate: pharmacology, mechanisms, and prevention. plate neurons in the formation of thalamocortical connections. Nature J Neurosci 13:1441–1453. 347:179 –181. Parent JM, Tada E, Fike JR, Lowenstein DH (1999) Inhibition of dentate gran- Goodman CS, Shatz CJ (1993) Developmental mechanisms that generate ule cell neurogenesis with brain irradiation does not prevent seizure-induced precise patterns of neuronal connectivity. Cell [Suppl] 72:77–98. mossy fiber synaptic reorganization in the rat. J Neurosci 19:4508 – 4519. Hack M, Flannery DJ, Schluchter M, Cartar L, Borawski E, Klein N (2002) Peterson BS, Vohr B, Staib LH, Cannistraci CJ, Dolberg A, Schneider KC, Katz Outcomes in young adulthood for very-low-birth-weight infants. N Engl KH, Westerveld M, Sparrow S, Anderson AW, Duncan CC, Makuch RW, J Med 346:149 –157. Gore JC, Ment LR (2000) Regional brain volume abnormalities and Han BH, D’Costa A, Back SA, Parsadanian M, Patel S, Shah AR, Gidday JM, long-term cognitive outcome in preterm infants. JAMA 284:1939 –1947. Srinivasan A, Deshmukh M, Holtzman DM (2000) BDNF blocks caspase-3 Piecuch RE, Leonard CH, Cooper BA, Kilpatrick SJ, Schlueter MA, Sola A activation in neonatal hypoxia-ischemia. Neurobiol Dis 7:38 –53. (1997) Outcome of infants born at 24 –26 weeks’ gestation, II: neurode- Hitchcock PF, Hickey TL (1980) Prenatal development of the human lateral velopmental outcome. Obstet Gynecol 90:809 – 814. geniculate nucleus. J Comp Neurol 194:395– 411. Price DJ, Aslam S, Tasker L, Gillies K (1997) Fates of the earliest generated Huttenlocher PR, de Courten C, Garey LJ, Van der Loos H (1982) Synapto- cells in the developing murine neocortex. J Comp Neurol 377:414 – 422. genesis in human visual cortex: evidence for synapse elimination during Rice JED, Vannucci RC, Brierley JB (1981) The influence of immaturity on normal development. Neurosci Lett 33:247–252. hypoxic-ischemic brain damage in the rat. Ann Neurol 9:131–141. Inder TE, Huppi PS, Warfield S, Kikinis R, Zientara GP, Barnes PD, Jolesz F, Sheldon RA, Chuai J, Ferriero DM (1996) A rat model for hypoxic-ischemic Volpe JJ (1999) Periventricular white matter injury in the premature brain damage in very premature infants. Biol Neonate 69:327–341. infant is followed by reduced cerebral cortical gray matter volume at term. Sheldon RA, Sedik C, Ferriero DM (1998) Strain-related brain injury in Ann Neurol 46:755–760. neonatal mice subjected to hypoxia-ischemia. Brain Res 810:114 –122. Jensen F (2002) The role of glutamate receptor maturation in perinatal sei- Spreafico R, Frassoni C, Arcelli P, Selvaggio M, De Biasi S (1995) In situ zures and brain injury. Int J Dev Neurosci 20:339 –347. labeling of apoptotic cell death in the cerebral cortex and thalamus of rats Johnston MV (1998) Selective vulnerability in the neonatal brain. Ann Neu- during development. J Comp Neurol 363:281–295. rol 44:155–156. Towfighi J, Mauger D, Vannucci RC, Vannucci SJ (1997) Influence of age on Johnston MV, Trescher WH, Ishida A, Nakajima W (2001) Neurobiology of the cerebral lesions in an immature rat model of cerebral hypoxia-ischemia: a hypoxic-ischemic injury in the developing brain. Pediatr Res 49:735–741. light microscopic study. Brain Res Dev Brain Res 100:149 –160. Kostovic I, Rakic P (1990) Developmental history of the transient subplate Vannucci RC (1990) Experimental biology of cerebral hypoxia-ischemia: zone in the visual and somatosensory cortex of the macaque monkey and relation to perinatal brain damage. Pediatr Res 27:317–326. human brain. J Comp Neurol 297:441– 470. Vitalis T, Cases O, Callebert J, Launay JM, Price DJ, Seif I, Gaspar P (1998) Kostovic I, Judas M, Rados M, Hrabac P (2002) Laminar organization of the Effects of monoamine oxidase A inhibition on barrel formation in the human fetal cerebrum revealed by histochemical markers and magnetic mouse somatosensory cortex: determination of a sensitive developmental resonance imaging. Cereb Cortex 12:536 –544. period. J Comp Neurol 393:169 –184. Lanzi G, Fazzi E, Uggetti C, Cavallini A, Danova S, Egitto MG, Ginevra OF, Volpe JJ (2001a) Neurobiology of periventricular leukomalacia in the pre- Salati R, Bianchi PE (1998) Cerebral visual impairment in periventricu- mature infant. Pediatr Res 50:553–562. lar leukomalacia. Neuropediatrics 29:145–150. Volpe JJ (2001b) Neurology of the newborn, Ed 4. Philadelphia: Saunders. Lein ES, Finney EM, McQuillen PS, Shatz CJ (1999) Subplate neuron abla- Wood NS, Marlow N, Costeloe K, Gibson AT, Wilkinson AR (2000) Neu- tion alters neurotrophin expression and ocular dominance column for- rologic and developmental disability after extremely preterm birth. EPI- mation. Proc Natl Acad Sci USA 96:13491–13495. Cure Study Group. N Engl J Med 343:378 –384. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Neuroscience Unpaywall

Selective Vulnerability of Subplate Neurons after Early Neonatal Hypoxia-Ischemia

McQuillen, Patrick S.; Ann Sheldon, R.; Shatz, Carla J.; Ferriero, Donna M.
Journal of Neuroscience – Apr 15, 2003
Download PDF
8 pages

Loading next page...
 
/lp/unpaywall/selective-vulnerability-of-subplate-neurons-after-early-neonatal-w1mLkMF388

References

References for this paper are not available at this time. We will be adding them shortly, thank you for your patience.

Publisher
Unpaywall
ISSN
0270-6474
DOI
10.1523/jneurosci.23-08-03308.2003
Publisher site
See Article on Publisher Site

Abstract

3308 • The Journal of Neuroscience, April 15, 2003 • 23(8):3308 –3315 Selective Vulnerability of Subplate Neurons after Early Neonatal Hypoxia-Ischemia 1,2 1 2 3 Patrick S. McQuillen, R. Ann Sheldon, Carla J. Shatz, and Donna M. Ferriero 1 2 3 Departments of Pediatrics and Neurology, University of California San Francisco Medical Center, San Francisco, California 94143-0106, and Department of Neurobiology, Harvard Medical School, Boston, Massachusetts 02115 Neonatal hypoxia-ischemia in the preterm human leads to selective injury to the subcortical developing white matter, which results in periventricular leukomalacia (PVL), a condition associated with abnormal neurodevelopment. Maturation-dependent vulnerability of late oligodendrocyte progenitors is thought to account for the cellular basis of this condition. A high frequency of cognitive and sensory deficits with decreasing gestational age suggests pervasive abnormalities of cortical development. In a neonatal rat model of hypoxic- ischemic injury that produces the characteristic pattern of subcortical injury associated with human PVL, selective subplate neuron death is seen. The premature subplate neuron death occurs after thalamic axons have reached their targets in cortex. Thus, as expected, thalamocortical connections form normally, including patterned connections to somatosensory cortex. However, deficits in motor function still occur, as in babies with PVL. Subplate neuron cell death in PVL provides another mechanism for abnormal neurodevelop- ment after neonatal hypoxia-ischemia. Key words: periventricular leukomalacia; visual; cortex; development; premature infant; thalamocortical creasing gestational age (Piecuch et al., 1997). Cortical visual im- Introduction pairment (i.e., visual loss caused by impairment of posterior vi- Hypoxia-ischemia (H-I) results in selective damage to different sual pathways) is particularly common, especially in infants with brain structures depending on the developmental stage at which PVL, in whom estimates range from 66% (Lanzi et al., 1998) to it occurs (Johnston, 1998). H-I in the preterm human [gestation- 94% in infants with severe PVL (Cioni et al., 1997). al week (GW) 23–32] causes damage to subcortical developing Subplate neurons are required for normal visual cortical de- white matter, a condition known as periventricular leukomalacia velopment (for review, see Allendoerfer and Shatz, 1994). They (PVL) (Volpe, 2001b). Developmental immaturity of the cerebral are among the first generated cells of neocortex (Luskin and vasculature is thought to account for this characteristic subcorti- Shatz, 1985) and come to lie beneath the developing cortical cal distribution (Volpe, 2001a). Other mechanisms include selec- plate, where they participate in the earliest neocortical circuitry tive cellular vulnerability, which relates to intrinsic properties of (Friauf and Shatz, 1991). Subplate neurons undergo pro- the vulnerable cell type. Cells of the oligodendrocyte lineage grammed cell death and are primarily absent from adult neocor- manifest stage-specific vulnerability to H-I (Back et al., 2002) tex (Chun et al., 1987). In humans, the subplate zone peaks in size through mechanisms of oxidative stress (Oka et al., 1993; Back et at GW 24, when the subplate is four times the width of the devel- al., 1998) and excitotoxicity (Matute et al., 1997; Fern and Moller, oping cortical plate (Kostovic and Rakic, 1990), and declines 2000; Follett et al., 2000). Subplate neurons, a transient cell type thereafter. The peak of subplate development coincides with the located beneath the cortical plate (Chun et al., 1987), are also gestational age associated with the highest incidence of PVL. vulnerable to stage-specific excitotoxicity (Chun and Shatz, Given their critical role in normal visual cortical development, 1988). the death of subplate neurons after H-I would illuminate mech- H-I in the preterm infant disrupts normal development and anisms that account for the high incidence of cortical visual im- results in significant cerebral injury. Neurological disability is pairment observed in PVL and provide a general model for ab- observed in 51% of premature infants (GW 25 weeks) exam- normal cortical development after H-I. In the present study, we ined at 30 months of age (Wood et al., 2000) and persists into examine a neonatal rodent model of H-I brain injury (Sheldon et adulthood (Hack et al., 2002). Deficits are found in motor, per- al., 1998) to determine whether subplate neurons die after neo- ceptual, and cognitive systems (Volpe, 2001b). These widespread natal H-I. abnormalities of cerebral development can be measured quanti- tatively with advanced magnetic resonance imaging (Miller et al., 2002). Cognitive impairment is associated significantly with de- Materials and Methods Animals. Timed-pregnant Sprague Dawley rats (Simonsen, Gilroy, CA) were allowed food and water ad libitum. All animal research was ap- Received Oct. 24, 2002; revised Jan. 24, 2003; accepted Jan. 29, 2003. proved by the University of California San Francisco Committee on An- This research was supported by National Eye Institute Grants EY02858 (C.J.S.) and NS35902 (D.M.F.) and National imal Research and was performed in accordance with standards of hu- Institutes of Health Grant K08 HD01396-03 (P.S.M.). We thank Cynthia Cowdrey for preparing cryostat sections and mane care set forth in the Policy on Humane Care and Use of Laboratory Michael DeFreitas and Gabriel Zada for assistance with bromodeoxyuridine, in situ end labeling, and double staining. Animals. Correspondence should be addressed to Dr. P. S. McQuillen, Department of Pediatrics, Box 0106, University of Hypoxia-ischemia. The manipulation was performed at postnatal day 1 California San Francisco Medical Center, San Francisco, CA 94143-0106. E-mail: [email protected]. Copyright © 2003 Society for Neuroscience 0270-6474/03/233308-08$15.00/0 (P1) or P2 (day of birth  P0) as described previously (Sheldon et al., McQuillen et al. • Subplate Neurotoxicity after Hypoxia-Ischemia J. Neurosci., April 15, 2003 • 23(8):3308 –3315 • 3309 1996). The first three litters received H-I at P1, and mortality was 31% ously (Parent et al., 1999). Primary antibody dilutions used were 1:200 (10 of 32). These animals were used for in situ end labeling (ISEL) and for anti-GFAP (G-A-5; Sigma) and 1:1000 for BrdU (mouse monoclonal; bromodeoxyuridine (BrdU) staining (Figs. 1–3). Because mortality was Roche Molecular Biochemicals). Primary antibody was applied over- lower (19%), and the pattern of injury by ISEL staining was unchanged, night at room temperature. Secondary antibody (biotinylated donkey subsequent litters received H-I at P2. These animals were used for anal- anti-mouse; Jackson ImmunoResearch) was applied for 1 hr at room ysis of thalamocortical innervation and motor testing (Figs. 4, 5). Total temperature. The sections were washed and developed with a mouse Elite mortality was 25% (16 of 64). To produce ischemia, pups were anesthe- ABC kit (Vector Laboratories, Burlingame, CA). tized with nitrous oxide, halothane, and oxygen. A midline incision was BrdU counting. The subplate zone was identified as described previ- made in the neck; the right common carotid artery was dissected from the ously (McQuillen et al., 2002) by accepted criteria (Boulder Committee, jugular vein and permanently coagulated with a bipolar coagulator 1970) and cytoarchitectonic features of neocortex (Bayer and Altman, [common carotid ligation (CCL)]. Sham-operated animals received the 1990). Specifically, in the radial domain, the subplate (layer VIB) was same operation, except that the carotid artery was not coagulated. Ani- localized at the base of the cortical plate, immediately below layer VI mals were returned to the dam for 1–2 hr. Subsequently, pups receiving neurons, and contained characteristic pyramidal neurons. The borders hypoxia were placed in 5.6% oxygen in chambers floating in a 37°C water in the coronal plane were determined by the characteristic six-layered bath for 3 hr. One pup from each litter was monitored with a skin surface neocortex and extended to cingulate cortex in the medial direction and temperature probe to ensure consistency between litters and that the entorhinal cortex laterally. Only heavily BrdU-labeled cells that fell into animals did not overheat. The body surface temperature was kept con- this region were counted. As in previous studies (Price et al., 1997), stant at 34°C (normal P7 core temperature, 35–37°C). Six litters of ro- heavily labeled cells were defined as cells in which more than half the dents, representing 64 animals, were used for the study. Rats were killed nucleus was labeled. Littermates were analyzed for quantification of sub- with pentobarbital, 100 mg/kg, given by intraperitoneal injection. plate neuron death. Brains from animals receiving H-I at P1 (n  6), Subplate neuron BrdU birth dating. Timed-pregnant Sprague Dawley sham-operated animals (n 3), or unmanipulated animals (n 2) were rats received an intraperitoneal injection of BrdU, 30 mg/kg (Sigma, St. analyzed at P5. Despite the use of timed-pregnant rats, significant vari- Louis, MO), at embryonic day 12.5 (E12.5; plug date, E0.5) to label ability occurs between litters in BrdU uptake and labeling of subplate subplate neurons in neocortex (Bayer and Altman, 1990). neurons. Therefore, no attempt was made to compare absolute numbers Fluorescent BrdU staining. BrdU-labeled subplate neurons were visu- of BrdU-positive neurons among experimental groups. Cell counts were alized as described previously (McQuillen et al., 2002). Brains were re- expressed as the interhemispheric ratio of BrdU-labeled subplate neu- moved rapidly from the cranium and flash-frozen in Tissue-Tek O.C.T. rons (BrdU-positive cells ipsilateral to CCL/BrdU-positive cells con- compound (Sakura Finetek, Torrance, CA) in a dry ice–95% ethanol tralateral to CCL). Cell death is expressed as a percentage derived from bath. Coronal cryostat sections (10 m thick) were fixed in 0.1 M sodium this ratio (100  ipsilateral/contralateral  100). The brains were sec- phosphate-buffered 4% paraformaldehyde, extracted with 0.6% Triton tioned entirely, and a random series representing every 10th section was X-100, acetylated, quenched in 3% hydrogen peroxide, and dehydrated selected and analyzed. There were no differences between groups in the through graded alcohols. To expose incorporated BrdU, the sections number of sections per animal (85  8.4 vs 82  8.4 sections per brain, were microwaved for 10 min in 0.1 M sodium citrate, pH 5.0. Anti-BrdU mean  SD; p  0.57); therefore, average cell death per section is re- antibody (IU4; Caltag, Burlingame, CA) was applied at 1:20,000 with ported. The subplate was identified as described above in digital images, exonuclease III (ExoIII) 100 U/l (Roche Molecular Biochemicals, Indi- and heavily labeled BrdU-positive cells were counted in each hemisphere anapolis, IN) in ExoIII buffer supplemented to 100 mM NaCl with 1% in each section with the cell-counting macro (ftp://rsbweb.nih.gov/pub/ bovine serum albumin at 37°C for 1 hr. After washing, horseradish nih-image/user-macros/). peroxidase-conjugated goat anti-mouse secondary antibody (Jackson Perl’s iron stain. Free-floating sections were stained in a 1:1 mixture of ImmunoResearch, West Grove, PA) was applied at 1:200 in blocking 2% v/v HCl and 2% potassium ferrocyanide for 30 min at room temper- solution (supplied by the manufacturer) for 30 min, followed by tyra- ature (Connor et al., 1995). The sections were rinsed in distilled water mide signal amplification (TSA) (Direct-green; PerkinElmer Life Sci- twice for 10 min, and then precipitated iron was visualized with DAB, 0.5 ences, Boston, MA). The sections were counterstained with 0.001% mg/ml, in 0.1 M phosphate buffer with 0.07% hydrogen peroxide for 5–10 bis-benzimide. min. The sections were rinsed in 0.1 M PBS and mounted on slides, ISEL staining. To visualize dying cells, we used a modified version of dehydrated, and put under cover glass. the ISEL method (Blaschke et al., 1996), performed as described pre- Cytochrome oxidase staining. Free-floating sections were stained for viously (McQuillen et al., 2002). A reaction mixture containing 1 M cytochrome oxidase activity (Anderson et al., 1975) in a reaction mixture biotin-deoxyuridine 5-triphosphate (Roche Molecular Biochemicals), that contained 5 mg of DAB, 10 mg of cytochrome C (Sigma), 750 mg of 0.15 U/ml terminal transferase (Invitrogen, Rockville, MD), 1 terminal sucrose, 20 mg of catalase (Sigma), and 9 ml of 0.05 M sodium phosphate transferase buffer, and 1% bovine serum albumin was applied, and the buffer, pH 7.4. Sections were incubated overnight at 37°C, rinsed in 0.1 M sections were incubated for 1 hr at 37°C. Sections were washed and then PBS, mounted on slides, dehydrated, and put under cover glass. incubated with NeutraLite avidin– horseradish peroxidase (Molecular Carbocyanine dye labeling. Rats were fixed by transcardial perfusion Probes, Eugene, OR) at 1:1000 in blocking solution (supplied with the with 0.1 M sodium phosphate-buffered 4% paraformaldehyde or TSA Direct kit) for 30 min. The sections were washed and developed with immersion-fixed in the same fixative (embryonic ages). Brains were re- TSA Direct-Cy3 (PerkinElmer Life Sciences). Double labeling was per- moved and stored in fixative with 0.02% sodium azide. Small (100 formed with ISEL reacted before microwave treatment and BrdU pri- m), similar-sized crystals of DiI (D-282; Molecular Probes) or 1,1- mary antibody incubation. Visualization with direct TSA was performed dioctadecyl-3,3,3,3-tetramethylindodicarbocyanine perchlorate (DiD; sequentially, with inactivation of peroxidase between development of D-307; Molecular Probes) were placed into visual cortex. Alternate con- ISEL and BrdU immunohistochemistry. Sections of neonatal rat thymus figurations of dyes were placed in each hemisphere to distinguish ipsilat- (positive) and liver (negative) were analyzed as controls for the sensitivity eral from contralateral hemispheres. The dye was allowed to transport at of ISEL staining. 37°C for 3 weeks. Coronal sections were cut at 50 –100 monavi- DAB immunohistochemistry. Animals were perfused transcardially bratome. Sections were counterstained with 0.001% bis-benzimide. with 0.1 M phosphate buffer followed by cold 4% paraformaldehyde/0.1 Imaging. Digital images were acquired with a Nikon (Mellville, NY) M phosphate buffer. Perfused brains were cryoprotected in 25% sucrose Eclipse 800 microscope and a cooled CCD camera (Spot2; Diagnostic in 0.1 M phosphate buffer before being sectioned on a sliding microtome. Instruments, Sterling Heights, MI). Digital images were analyzed on an Fifty micrometer sections were quenched with 3% hydrogen peroxide, Apple (Cupertino, CA) G4 computer with the public domain NIH Image washed, and blocked (5% horse serum, 5% fish skin gelatin, and 0.1% program (developed at the National Institutes of Health and available on Triton X-100 in 0.1 M Tris-buffered saline). For BrdU staining, DNA was the Internet at http://rsb.info.nih.gov/nih-image/). denatured by incubating sections in 2N HCl at 37°C for 30 min and then Confocal montage imaging. Fluorescent cryosections (BrdU/ISEL) washed in 0.1 M sodium borate, pH 8.5, for 10 min as described previ- were imaged on a Leica (Cambridge, UK) confocal microscope. Low- 3310 • J. Neurosci., April 15, 2003 • 23(8):3308 –3315 McQuillen et al. • Subplate Neurotoxicity after Hypoxia-Ischemia magnification montages were reconstructed from 5 or 10 fields with Leica Qwin montaging software and a motorized stage. Complete sec- tions represent a montage of 8 –10 separate fields. Motor testing. Animals were raised to 3 months of age and then as- sessed for motor deficits (Crawley, 2000). Each animal was observed for gross deficits with open-field locomotion. Then animals were scored on a three-point scale (2, normal; 1, impaired; and 0, incapable of performing task) for rod and beam walking (1-inch-diameter rod, 1-inch-wide beam) and for the number of foot faults on stair climbing. Each animal was tested three times on each test, and the median value was analyzed for significance. Finally, gait analysis was performed with footprint pattern. Animals receiving H-I were compared with controls consisting of both unmanipulated littermates and sham-operated animals. Statistics. Normally distributed data are reported as mean  SD. Hy- pothesis testing for differences between groups was performed with an unpaired, two-tailed t test. Nonparametric data and percents are re- ported as mean  interquartile range (subplate cell death) or mean of median scores (motor testing) SEM. Differences between groups were determined with Mann–Whitney U test and corrected for ties. Results P1 rodent H-I results in subcortical, periventricular cell death To determine the pattern of cell death after CCL and hypoxia at P1 (H-I), we used fluorescent ISEL for detection of DNA strand breaks associated with dying cells, combined with a nuclear coun- terstain to visualize anatomy. After H-I, cells may die with mor- phologic features of necrosis or apoptosis (Northington et al., 2001). We use ISEL staining solely as a sensitive indicator of cell death, with the knowledge that cells dying with either phenotype may display an ISEL signal (Charriaut-Marlangue and Ben-Ari, 1995). DNA fragmentation can be detected normally in the brains of postnatal rats (Spreafico et al., 1995) and represents naturally occurring cell death during development. This normal pattern of cell death can be appreciated in sham-operated and unmanipulated littermates (data not shown) in the same pattern observed in the hemisphere contralateral to the carotid ligation in this model (Fig. 1 A–C, right hemispheres). As has been noted previously (Vannucci, 1990), hypoxia or ischemia alone does not result in any detectable increase in cell death. Superimposed on this naturally occurring cell death in the hemisphere ipsilateral to CCL is cell death that resulted from H-I (Fig. 1 A–C, left hemi- spheres). At 12 hr after manipulation, cell death caused by H-I peaked, and a broad band of cell death could be appreciated in the subcortical regions that contained subplate, intermediate, and subventricular zones (Fig. 1 B,C). The ventricular zone was af- fected but to a lesser degree. A smaller amount of cell death was also apparent scattered throughout the neocortex, and retrosple- nial cortex was especially affected (Fig. 1 B, asterisk). Within the thalamus, the reticular nucleus and internal capsule showed in- creased cell death (Fig. 1 B, arrowheads). In the hippocampus, dying cells were not observed in the pyramidal layer in any hip- pocampal subdivision. Scattered dying cells were noted in the hippocampal subgranular layer in both hemispheres. This pat- tern of cell death was present throughout the rostral-caudal ex- tent of cerebral cortex, beginning at the anterior commissure and extending into occipital cortex (Fig. 1 A–C). Cell death in the intermediate zone was most intense in parietal cortex at the level Figure 1. Subcortical topography of cell death 12 hr after P1 H-I. Dying cells with DNA strand of thalamus but extended to more caudal brain regions, including breaks are detected with ISEL 12 hr after H-I at P1. A–C, ISEL signal (red) and bis-benzimide visual and visual association areas, following the dorsolateral as- nuclear counterstain (blue) are shown alone in coronal sections from frontal ( A), parietal ( B), pect of the lateral ventricles (Fig. 1 A–C). Temporally, ISEL attrib- and occipital ( C) regions. Arrowheads mark cell death in reticular thalamus. Asterisk marks cell utable to H-I could be detected as early as the end of the hypoxic death in retrosplenial cortex. period in animals that died during the procedure (data not shown). By 12 hr after hypoxia, the ISEL signal peaked (Fig. 1); it diminished by 24 hr (Fig. 2), and by 4 d after H-I, increased cell McQuillen et al. • Subplate Neurotoxicity after Hypoxia-Ischemia J. Neurosci., April 15, 2003 • 23(8):3308 –3315 • 3311 (compare Fig. 2, compare C, D). There was a corresponding increase in ISEL- labeled dying cells in the subplate and in- termediate zone ipsilateral to CCL (Fig. 2 A,C), although the amount of cell death was less than at 12 hr after manipulation (Fig. 1). Using BrdU immunohistochem- istry in combination with ISEL labeling, we identified double-labeled cells that indi- cated dying subplate neurons (Fig. 2 B,C, vertical arrows). These cells were noted fre- quently only in the hemispheres receiving H-I. In animals receiving H-I in which dou- ble labeling was performed (n  4), we found significantly more double-labeled dy- ing subplate neurons ipsilateral to CCL than contralateral (4.5 1.9 vs 0.4 0.5 cells per section; p 0.0001). Quantification of subplate neuron cell death after H-I There is significant interanimal variability in the amount of damage after neonatal H-I (Rice et al., 1981; Sheldon et al., 1996, 1998; Towfighi et al., 1997). To quantify the extent and variability of subplate neu- Figure 2. Cell death and BrdU staining for subplate neurons 24 hr after P1 H-I. ISEL (red), BrdU immunohistochemistry (green), ron cell death after H-I, we examined and bis-benzimide nuclear counterstain (blue) in coronal sections taken from parietal cortex 24 hr after H-I at P1. A, Low- BrdU-positive cell density 4 d after H-I at magnification views showing both hemispheres. Large white boxes in A delineate higher-magnification views in D of contralateral hemisphere (hypoxia) and in C of ipsilateral hemisphere (H-I), and small white box indicates region shown in B that demonstrated P1 using systematic random sampling and single ISEL-positive (horizontal arrow), BrdU-positive (asterisk), and double-positive (vertical arrow) cells. SP, Subplate. digital imaging to quantify subplate neu- ron cell death per section (see Materials and Methods). Subplate neuron cell death death was not detectable (data not shown). Cell death was not was increased significantly by H-I (45  48 vs 2  9%, mean assayed at time points beyond 96 hr. interquartile range; p  0.006, Mann–Whitney U test; Fig. 3A). To confirm this pattern of subcortical injury with other methods, The severity of injury in any given animal was readily apparent we performed GFAP immunohistochemistry and Perl’s iron stain- from inspection of either the BrdU labeling or cresyl violet stain- ing 4 d after H-I at P1 (data not shown). Both GFAP immunohisto- ing and ranged from mildly increased death (Fig. 3D) to nearly chemistry and iron staining were increased in the subplate and in- complete death of heavily BrdU-labeled subplate neurons (Fig. termediate zones in exactly the distribution of the heaviest ISEL 3B). The variable neuronal injury that resulted from this manip- staining. Cresyl violet staining of neocortex was remarkably normal ulation, with injury to subplate neurons even in the mildest cases, despite the presence of scattered ISEL, and the histologic appearance confirms the selective vulnerability of subplate neurons relative of cortex did not reveal prominent cell loss (Fig. 3B–D). Animals to other neuronal populations at this age. with severe injury did show some loss of lower-layer neurons in neocortex (Fig. 3B). Thalamocortical connections form normally after neonatal H-I Subplate neurons die after neonatal H-I BrdU labeling suggests that significant numbers of subplate neu- To determine whether subplate neurons specifically were among rons die prematurely after neonatal H-I. To determine the effects dying cells, we permanently labeled subplate neurons as they of this premature subplate neuron cell death on thalamocortical were generating from dividing neuroblasts with a labeled nucle- development, it was first necessary to determine whether otide, BrdU (BrdU birth dating). Subplate neurons are generated thalamocortical connections formed normally. Given the signif- from dividing neuroblasts from E10.5 to E12.5 (Bayer and Alt- icant cell death in the intermediate and subventricular zones, it is man, 1990). A pulse of BrdU is taken up by dividing neuroblasts possible that there was significant injury to the developing in S-phase. Only neurons generated from the next round of cell thalamocortical and corticofugal axons. Excitotoxic ablation of division (i.e., subplate and marginal zone neurons) receive heavy subplate neurons in cat, before ingrowth of thalamic axons into BrdU label. The BrdU is diluted with each successive round of cell visual cortex, prevents normal innervation of their targets in layer division, so that later-generated neurons (e.g., layer VI-II) receive IV (Ghosh et al., 1990). However, ablation immediately after progressively lighter label. Immunohistochemistry can then de- innervation does not result in the absence of thalamic innerva- tect BrdU in heavily labeled subplate neurons at any age. BrdU tion of layer IV (Ghosh and Shatz, 1992). In rat, thalamic axons birth dating is currently the optimal method for identifying sub- have reached neocortex by P0 and innervate appropriate targets plate neurons (Allendoerfer and Shatz, 1994). over the subsequent days (Catalano et al., 1996). On the basis of At 24 hr after H-I, a comparison of BrdU staining in the hemi- these observations, we predicted that H-I at P2 would not disrupt sphere ipsilateral to CCL with the contralateral hemisphere visual thalamocortical innervation. Indeed, this is what we ob- showed a significant decrease in BrdU-labeled subplate neurons served using the lipophilic carbocyanine dyes DiI and DiD to 3312 • J. Neurosci., April 15, 2003 • 23(8):3308 –3315 McQuillen et al. • Subplate Neurotoxicity after Hypoxia-Ischemia trace thalamocortical connections at P7 after H-I at P2. At these ages, label in thal- amus resulting from dye placement in cortex is a combination of retrograde la- beling of thalamic neurons and antero- grade labeling of the descending cortico- thalamic projections. After dye crystal placement into visual cortex (n 5), there was robust label in the lateral geniculate nucleus (visual thalamus) in both hemi- spheres (Fig. 4C,D). Furthermore, despite extensive cell death observed in the inter- nal capsule (Fig. 1 B), labeled fibers could be observed coursing normally through the intermediate zone and turning medi- ally into the internal capsule before enter- ing thalamus (Fig. 4 A,B). Ablation of subplate neurons immedi- ately after innervation of layer IV prevents the activity-dependent refinement of thalamocortical connections into ocular dominance columns (Ghosh and Shatz, 1992). Rodent visual cortex contains monocular and binocular zones but no finer organization of ocular dominance (Antonini et al., 1999). To assess the de- velopment of patterned thalamocortical Figure 3. Quantification of BrdU-positive subplate neurons 4 d after H-I at P1. A, Plot of subplate neuron cell death (see connections after H-I at P2, we visualized Materials and Methods) comparing animals receiving H-I with controls (sham-operated and unmanipulated littermate; p the topographical whisker barrel repre- 0.006). Severely ( B), moderately ( C), and mildly ( D) affected examples of subplate neuron BrdU immunohistochemistry and cresyl violet staining from sections used for quantification of subplate neuron death are shown. Plotted percent cell death for each sentation in somatosensory cortex at P10. example (B, C, D) is indicated by letter in A. SP, Subplate. The whisker barrel representation is con- solidated over the first postnatal week we observed faults of the left rear paw most frequently in animals (O’Leary et al., 1994), and genetic manipulations of glutamater- receiving right CCL and hypoxia. These observations indicate that gic (Cases et al., 1996) and serotonergic (Vitalis et al., 1998) neu- despite normal-appearing open-field locomotion, P2 H-I leads to rotransmission disrupt barrel formation. In every case we exam- significant motor deficits. The relationship of these motor deficits to ined at P10 (n  4), after H-I at P2, well formed cytochrome subplate cell death remains to be elucidated. oxidase patches appeared in layer IV of somatosensory neocortex ipsilateral to CCL (Fig. 4 E) that were identical to those observed in Discussion the contralateral hemisphere (Fig. 4 F). We conclude from these analyses that P2 H-I does not disrupt These results demonstrate that a rat model of early neonatal H-I initial thalamocortical pathfinding and innervation of somato- leads to significant, premature subplate neuron cell death. De- sensory and visual cortex. Moreover, the initial development of spite intense subcortical injury to the developing subplate and patterned connections in somatosensory cortex proceeds nor- intermediate zones, thalamocortical connections to somatosen- mally. Our analysis does not address subsequent refinement and sory and visual cortex form normally. This manipulation results plasticity of sensory thalamocortical connections. in measurable motor deficits in mature animals. These data, combined with observations of injury to oligodendrocyte pro- P2 H-I results in motor deficits genitors (Back et al., 2002), support H-I at P2 in rat as a model of Human PVL is characterized by dysmyelination and spastic di- human PVL. This model is useful for testing the hypothesis that plegia, a static motor deficit with more pronounced involvement neonatal H-I disrupts neurodevelopment through effects on cor- of lower extremities than of upper extremities (Volpe, 2001b). To tical plasticity and the refinement of cortical connections as a determine whether H-I at P2 leads to motor deficits, we allowed result of subplate neuron cell death. animals from two litters to develop to maturity after H-I at P2. Animals were then observed during open-field locomotion and Maturation-dependent topography of H-I cerebral injury gait analysis, but they did not display any observable deficits (data H-I brain injury produces age-dependent and region-specific in- not shown). However, when tested on rod and beam walking, jury. Many factors contribute to the evolution of this pattern, animals receiving H-I performed significantly worse than controls including maturation of cerebral oxygen and substrate delivery (Fig. 5; rod walking score, 1.3 0.2vs0.7 0.1, control vs H-I, mean through development of the cerebral vasculature and autoregu- of median scores  SEM; tied p  0.04, Mann–Whitney U test; lation (Volpe, 2001a), selective cellular vulnerability related to beam walking score, 1.8  0.1 vs 1.3  0.1; tied p  0.02). To oxidative metabolism (Ferriero, 2001), excitatory amino acid sig- confirm this observation, rats were tested for foot faults while they naling (Johnston et al., 2001; Jensen, 2002), and programmed cell climbed an inclined stair. H-I-treated animals had more faults than death (Han et al., 2000; Northington et al., 2001). The Levine controls (Fig. 5; 2.4 0.3 vs 0.2 0.1; p 0.0001, Mann–Whitney model [i.e., right CCL followed by hypoxia (Levine, 1960)] per- U test). Although faults were not recorded by extremity, qualitatively formed on immature rats (Rice et al., 1981) at selected ages (Shel- McQuillen et al. • Subplate Neurotoxicity after Hypoxia-Ischemia J. Neurosci., April 15, 2003 • 23(8):3308 –3315 • 3313 Figure 4. Thalamocortical projection to somatosensory and visual cortex after P2 H-I. A–D, Thalamocortical and corticothalamic axons labeled with DiI and DiD crystals placed in auditory or visual cortex. Coronal sections at the level of internal capsule (A, B) or lateral geniculate nucleus (C, D) demonstrate labeled fibers and cell bodies. To distinguish ipsilateral/H-I hemi- sphere from contralateral/hypoxic hemisphere, the dyes were placed in opposite configuration. For this example, the ipsilateral hemisphere (A, C) had DiI (red) placed in visual cortex and DiD (green) placed in auditory cortex. The contralateral hemisphere (B, D) had DiD (green) placed in visual cortex and DiI (red) placed in auditory cortex. Axons can be seen traversing the internal capsule (A, B), and neurons in lateral geniculate (visual thalamus) are back-labeled (C, D)inan identical manner in the two hemispheres. E, F, Cytochrome oxidase staining of sensory thalamo- cortical axons in patchy representation of whisker barrels in hemisphere receiving H-I ( E) and hypoxia alone ( F). don et al., 1996; Towfighi et al., 1997) also produces age- dependent and region-specific brain injury. Analyzing H-I at P1 with ISEL, we clearly show a subcortical pattern of cell death Figure 5. Motor deficits and abnormal myelination in mature animals after H-I at P2. Animals similar to human PVL. In agreement with others (Towfighi et al., receiving H-I (n 11) performed significantly worse on motor testing (rod walking, tied p 0.04; 1997), we did not see significant hippocampal injury at this age, a beam walking, tied p 0.02; and stair climbing, p 0.0001) than controls [unmanipulated litter- distinct difference from H-I at P7 and later, when hippocampal mate (n 8) and sham-operated (n 2)]. For rod and beam walking, score 2, normal; 1, impaired; region CA3 becomes vulnerable to injury before the adult pattern and 0, unable to perform task. For stair climbing, animals were scored for the number of foot slips of CA1 injury occurs beginning at P21. With this sensitive assay while climbing. Values are mean of the median score SEM. HI, Hypoxia-ischemia. for cell death, we did note low levels of diffuse cell death in neo- cortex and retrosplenial cortex. In the most severely affected an- imals, lower cortical layers begin to show laminar cell loss. In even ternatively, this could represent altered cortical development af- the mildest cases, subplate neuron cell death occurred, which ter injury to subcortical areas. confirms the selective vulnerability of these neurons relative to other neuronal populations. We did not note columnar patterns Selective cellular vulnerability: subplate neurons of cell death that are frequently observed at P7 or laminar injury Many authors have speculated that changing patterns of brain to cortical layers III and V as occurs at P13 (Towfighi et al., 1997). injury during development relate to intrinsic properties of the Most descriptions of the pathology of PVL indicate that neocor- affected cell type, i.e., selective cellular vulnerability (Mattson et tex is spared (Banker and Larroche, 1962). However, using sen- al., 1989; Johnston, 1998; Volpe, 2001a). Late oligodendrocyte sitive quantitative volumetric techniques with magnetic reso- progenitors are the prototypical examples of such a cell type, nance imaging of human infants with PVL, several studies (Inder because they manifest stage-specific vulnerability in vitro to exci- et al., 1999; Peterson et al., 2000) noted marked reduction in totoxicity (Matute et al., 1997; Fern and Moller, 2000), oxidative cerebral cortical gray matter volume throughout the brain, which stress (Oka et al., 1993; Back et al., 1998), and oxygen– glucose raises the possibility that neuronal cell death in neocortex may be deprivation (Fern and Moller, 2000) and in vivo to H-I (Back et more widespread than has been appreciated in human PVL. Al- al., 2002). In humans, preoligodendrocytes are the predominant 3314 • J. Neurosci., April 15, 2003 • 23(8):3308 –3315 McQuillen et al. • Subplate Neurotoxicity after Hypoxia-Ischemia cell type in the oligodendrocyte lineage during the developmental ruption of column formation after subplate neuron ablation is not period of peak incidence of PVL (Back et al., 2001). known, one possibility was suggested by the effects of subplate neu- Subplate neurons share many of these same properties. Sub- ron ablation on BDNF expression (Lein et al., 1999). Kainate injec- plate neurons are a transient cell population (Chun et al., 1987; tion leads to a long-lasting increase in BNDF expression localized to Al-Ghoul and Miller, 1989; Price et al., 1997), and they undergo the region of subplate ablation. Increased BDNF is associated with programmed cell death in the first postnatal week in mice (Mc- alterations in the phenotype of cortical inhibitory neurons, leading Quillen et al., 2002). Subplate neurons are located beneath the to the suggestion that subplate neurons modulate activity- developing neocortex (Luskin and Shatz, 1985; Kostovic et al., dependent competition by regulating levels of neurotrophins and 2002) near areas of diffuse white matter signal abnormality seen excitability in the developing cortex. on magnetic resonance imaging in preterm human infants Abnormal or delayed myelination is the hallmark of PVL. H-I at (Maalouf et al., 2001) at risk for the diffuse type of PVL (Volpe, P7 depletes the subventricular zone of oligodendrocyte progenitors 2001b). In humans, the subplate zone peaks at the onset of the (Levison et al., 2001). However, H-I at P2 and P7 results in the developmental window of vulnerability to PVL (GW 24) and proliferation of reactive late oligodendrocyte progenitors (Back et undergoes dissolution during the third trimester, and subplate al., 2002), and decreased myelin basic protein expression is only neurons are largely absent after 6 months of postnatal age (Kos- transient after H-I at P7 (Liu et al., 2002). Although cortical visual tovic and Rakic, 1990). Subplate neurons undergo programmed impairment is associated frequently with abnormalities on magnetic cell death to a much greater extent than other cortical neurons resonance imaging, 71% of premature infants with moderate PVL (Price et al., 1997). Subplate neurons are vulnerable to excito- during the neonatal period were found to have at least one abnor- toxic cell death (Chun and Shatz, 1988), which allows for the mality of visual testing at 1 year of age, and yet 66% of these children selective ablation of subplate neurons after injection of the gluta- had normal optic radiations, and all had normal-appearing visual mate agonist kainate into embryonic and postnatal kittens cortex (Cioni et al., 1997). These findings underscore the need to (Ghosh et al., 1990; Ghosh and Shatz, 1992). At later time points, examine fully the neurobiology of neonatal H-I and its impact on cortical neurons become sensitive to kainate, and the injections cortical development. Subplate neuron injury, alone or coincident no longer result in a selective ablation of subplate neurons with oligodendrocyte injury, could explain these observations. (Ghosh and Shatz, 1994). Subplate neurons become incorpo- References rated into mature synaptic networks, receiving excitatory input Al-Ghoul WM, Miller MW (1989) Transient expression of Alz-50 immuno- from thalamus and making excitatory connections with neurons reactivity in developing rat neocortex: a marker for naturally occurring in layer IV of neocortex, as well as sending recurrent collateral neuronal death? Brain Res 481:361–367. projections back to thalamus (Friauf and Shatz, 1991). In the Allendoerfer KL, Shatz CJ (1994) The subplate, a transient neocortical present study, we have demonstrated that P2 H-I leads to mod- structure: its role in the development of connections between thalamus erate to near-complete subplate neuron cell death, whereas most and cortex. Annu Rev Neurosci 17:185–218. Anderson WA, Bara G, Seligman AM (1975) The ultrastructural localization of cortical neurons are left intact. The mechanism of this selective cytochrome oxidase via cytochrome. J Histochem Cytochem 23:13–20. vulnerability of subplate neurons to H-I is unknown but may Antonini A, Fagiolini M, Stryker MP (1999) Anatomical correlates of func- relate to early cellular maturation (Chun and Shatz, 1989), with a tional plasticity in mouse visual cortex. J Neurosci 19:4388 – 4406. developmentally related increase in glutamate receptor expres- Back SA, Gan X, Li Y, Rosenberg PA, Volpe JJ (1998) Maturation- sion, including NMDA receptor 1 (Catalano et al., 1997) and dependent vulnerability of oligodendrocytes to oxidative stress-induced AMPA and kainate receptors (Furuta and Martin, 1999). Nota- death caused by glutathione depletion. J Neurosci 18:6241– 6253. bly, most of these observations have been made in vivo. We have Back SA, Luo NL, Borenstein NS, Levine JM, Volpe JJ, Kinney HC (2001) Late oligodendrocyte progenitors coincide with the developmental win- described recently a method for purifying subplate neurons that dow of vulnerability for human perinatal white matter injury. J Neurosci will be useful in elucidating cellular mechanisms of subplate neu- 21:1302–1312. ron cell death in vitro (DeFreitas et al., 2001). Back SA, Han BH, Luo NL, Chricton CA, Xanthoudakis S, Tam J, Arvin KL, Holtzman DM (2002) Selective vulnerability of late oligodendrocyte Abnormal cortical development after neonatal H-I: role of progenitors to hypoxia-ischemia. J Neurosci 22:455– 463. subplate neuron cell death Banker BQ, Larroche JC (1962) Periventricular leukomalacia of infancy. Arch Neurol 7:386 – 410. The involvement of subplate neurons in neonatal H-I brain in- Bayer SA, Altman J (1990) Development of layer I and the subplate in the rat jury is significant in light of the role that subplate neurons play in neocortex. Exp Neurol 107:48 – 62. normal cortical development (for review, see Allendoerfer and Blaschke AJ, Staley K, Chun J (1996) Widespread programmed cell death in Shatz, 1994). Human thalamocortical development begins at the proliferative and postmitotic regions of the fetal cerebral cortex. Devel- time of the development of layers in the lateral geniculate nucleus opment 122:1165–1174. at GW 22–25 (Hitchcock and Hickey, 1980). Synaptogenesis in Boulder Committee (1970) Embryonic vertebrate central nervous system: revised terminology. Anat Rec 166:257–261. human visual cortex occurs between GW 28 and birth (Hutten- Cases O, Vitalis T, Seif I, De Maeyer E, Sotelo C, Gaspar P (1996) Lack of barrels locher et al., 1982). In rodent, somatosensory thalamic afferents in the somatosensory cortex of monoamine oxidase A-deficient mice: role of have reached cortex at P0 (Catalano et al., 1996) and innervate a serotonin excess during the critical period. Neuron 16:297–307. their targets in layer IV soon thereafter. Thus, our model and Catalano SM, Robertson RT, Killackey HP (1996) Individual axon mor- human PVL occur at a stage of visual cortical development most phology and thalamocortical topography in developing rat somatosen- analogous to the early postnatal cat when thalamic afferents have sory cortex. J Comp Neurol 367:36 –53. entered visual cortex but have not segregated into ocular domi- Catalano SM, Chang CK, Shatz CJ (1997) Activity-dependent regulation of NMDAR1 immunoreactivity in the developing visual cortex. J Neurosci nance columns. Ablation at this point in development disrupts 17:8376 – 8390. the activity-dependent refinement of thalamocortical connections Charriaut-Marlangue C, Ben-Ari Y (1995) A cautionary note on the use of into mature ocular dominance columns (Ghosh and Shatz, 1992). the TUNEL stain to determine apoptosis. NeuroReport 7:61– 64. Ocular dominance columns form through an activity-dependent Chun JJ, Shatz CJ (1988) A fibronectin-like molecule is present in the devel- competition (for review, see Goodman and Shatz, 1993), possibly for oping cat cerebral cortex and is correlated with subplate neurons. J Cell neurotrophins. Although the cellular and molecular basis of the dis- Biol 106:857– 872. McQuillen et al. • Subplate Neurotoxicity after Hypoxia-Ischemia J. Neurosci., April 15, 2003 • 23(8):3308 –3315 • 3315 Chun JJ, Shatz CJ (1989) The earliest-generated neurons of the cat cerebral Levine S (1960) Anoxic-ischemic encephalopathy in rats. Am J Pathol cortex: characterization by MAP2 and neurotransmitter immunohisto- 36:139 –147. chemistry during fetal life. J Neurosci 9:1648 –1667. Levison SW, Rothstein RP, Romanko MJ, Snyder MJ, Meyers RL, Vannucci SJ (2001) Hypoxia/ischemia depletes the rat perinatal subventricular zone Chun JJ, Nakamura MJ, Shatz CJ (1987) Transient cells of the developing mammalian telencephalon are peptide-immunoreactive neurons. Nature of oligodendrocyte progenitors and neural stem cells. Dev Neurosci 23:234 –247. 325:617– 620. Cioni G, Fazzi B, Coluccini M, Bartalena L, Boldrini A, van Hof-van Duin J Liu Y, Silverstein FS, Skoff R, Barks JD (2002) Hypoxic-ischemic oligoden- droglial injury in neonatal rat brain. Pediatr Res 51:25–33. (1997) Cerebral visual impairment in preterm infants with periventricu- Luskin MB, Shatz CJ (1985) Studies of the earliest generated cells of the cat’s lar leukomalacia. Pediatr Neurol 17:331–338. visual cortex: cogeneration of subplate and marginal zones. J Neurosci Connor JR, Pavlick G, Karli D, Menzies SL, Palmer C (1995) A histochem- 5:1062–1075. ical study of iron-positive cells in the developing rat brain. J Comp Neurol Maalouf EF, Duggan PJ, Counsell SJ, Rutherford MA, Cowan F, Azzopardi D, 355:111–123. Edwards AD (2001) Comparison of findings on cranial ultrasound and Crawley JN (2000) What’s wrong with my mouse? Behavioral phenotyping magnetic resonance imaging in preterm infants. Pediatrics 107:719 –727. of transgenic and knockout mice. New York: Wiley-Liss. Mattson MP, Guthrie PB, Kater SB (1989) Intrinsic factors in the selective DeFreitas MF, McQuillen PS, Shatz CJ (2001) A novel p75NTR signaling vulnerability of hippocampal pyramidal neurons. Prog Clin Biol Res pathway promotes survival, not death, of immunopurified neocortical 317:333–351. subplate neurons. J Neurosci 21:5121–5129. Matute C, Sanchez-Gomez MV, Martinez-Millan L, Miledi R (1997) Gluta- Fern R, Moller T (2000) Rapid ischemic cell death in immature oligoden- mate receptor-mediated toxicity in optic nerve oligodendrocytes. Proc drocytes: a fatal glutamate release feedback loop. J Neurosci 20:34 – 42. Natl Acad Sci USA 94:8830 – 8835. Ferriero DM (2001) Oxidant mechanisms in neonatal hypoxia-ischemia. McQuillen PS, DeFreitas MF, Zada G, Shatz CJ (2002) A novel role for Dev Neurosci 23:198 –202. p75NTR in subplate growth cone complexity and visual thalamocortical Follett PL, Rosenberg PA, Volpe JJ, Jensen FE (2000) NBQX attenuates ex- innervation. J Neurosci 22:3580 –3593. citotoxic injury in developing white matter. J Neurosci 20:9235–9241. Miller SP, Vigneron DB, Henry RG, Bohland MA, Ceppi-Cozzio C, Hoffman Friauf E, Shatz CJ (1991) Changing patterns of synaptic input to subplate C, Newton N, Partridge JC, Ferriero DM, Barkovich AJ (2002) Serial and cortical plate during development of visual cortex. J Neurophysiol quantitative diffusion tensor MRI of the premature brain: development in 66:2059 –2071. newborns with and without injury. J Magn Reson Imaging 16:621– 632. Furuta A, Martin LJ (1999) Laminar segregation of the cortical plate during Northington FJ, Ferriero DM, Graham EM, Traystman RJ, Martin LJ (2001) corticogenesis is accompanied by changes in glutamate receptor expres- Early neurodegeneration after hypoxia-ischemia in neonatal rat is necro- sion. J Neurobiol 39:67– 80. sis while delayed neuronal death is apoptosis. Neurobiol Dis 8:207–219. Ghosh A, Shatz CJ (1992) Involvement of subplate neurons in the forma- O’Leary DD, Ruff NL, Dyck RH (1994) Development, critical period plas- tion of ocular dominance columns. Science 255:1441–1443. ticity, and adult reorganizations of mammalian somatosensory systems. Ghosh A, Shatz CJ (1994) Segregation of geniculocortical afferents during Curr Opin Neurobiol 4:535–544. the critical period: a role for subplate neurons. J Neurosci 14:3862–3880. Oka A, Belliveau MJ, Rosenberg PA, Volpe JJ (1993) Vulnerability of oligo- Ghosh A, Antonini A, McConnell SK, Shatz CJ (1990) Requirement for sub- dendroglia to glutamate: pharmacology, mechanisms, and prevention. plate neurons in the formation of thalamocortical connections. Nature J Neurosci 13:1441–1453. 347:179 –181. Parent JM, Tada E, Fike JR, Lowenstein DH (1999) Inhibition of dentate gran- Goodman CS, Shatz CJ (1993) Developmental mechanisms that generate ule cell neurogenesis with brain irradiation does not prevent seizure-induced precise patterns of neuronal connectivity. Cell [Suppl] 72:77–98. mossy fiber synaptic reorganization in the rat. J Neurosci 19:4508 – 4519. Hack M, Flannery DJ, Schluchter M, Cartar L, Borawski E, Klein N (2002) Peterson BS, Vohr B, Staib LH, Cannistraci CJ, Dolberg A, Schneider KC, Katz Outcomes in young adulthood for very-low-birth-weight infants. N Engl KH, Westerveld M, Sparrow S, Anderson AW, Duncan CC, Makuch RW, J Med 346:149 –157. Gore JC, Ment LR (2000) Regional brain volume abnormalities and Han BH, D’Costa A, Back SA, Parsadanian M, Patel S, Shah AR, Gidday JM, long-term cognitive outcome in preterm infants. JAMA 284:1939 –1947. Srinivasan A, Deshmukh M, Holtzman DM (2000) BDNF blocks caspase-3 Piecuch RE, Leonard CH, Cooper BA, Kilpatrick SJ, Schlueter MA, Sola A activation in neonatal hypoxia-ischemia. Neurobiol Dis 7:38 –53. (1997) Outcome of infants born at 24 –26 weeks’ gestation, II: neurode- Hitchcock PF, Hickey TL (1980) Prenatal development of the human lateral velopmental outcome. Obstet Gynecol 90:809 – 814. geniculate nucleus. J Comp Neurol 194:395– 411. Price DJ, Aslam S, Tasker L, Gillies K (1997) Fates of the earliest generated Huttenlocher PR, de Courten C, Garey LJ, Van der Loos H (1982) Synapto- cells in the developing murine neocortex. J Comp Neurol 377:414 – 422. genesis in human visual cortex: evidence for synapse elimination during Rice JED, Vannucci RC, Brierley JB (1981) The influence of immaturity on normal development. Neurosci Lett 33:247–252. hypoxic-ischemic brain damage in the rat. Ann Neurol 9:131–141. Inder TE, Huppi PS, Warfield S, Kikinis R, Zientara GP, Barnes PD, Jolesz F, Sheldon RA, Chuai J, Ferriero DM (1996) A rat model for hypoxic-ischemic Volpe JJ (1999) Periventricular white matter injury in the premature brain damage in very premature infants. Biol Neonate 69:327–341. infant is followed by reduced cerebral cortical gray matter volume at term. Sheldon RA, Sedik C, Ferriero DM (1998) Strain-related brain injury in Ann Neurol 46:755–760. neonatal mice subjected to hypoxia-ischemia. Brain Res 810:114 –122. Jensen F (2002) The role of glutamate receptor maturation in perinatal sei- Spreafico R, Frassoni C, Arcelli P, Selvaggio M, De Biasi S (1995) In situ zures and brain injury. Int J Dev Neurosci 20:339 –347. labeling of apoptotic cell death in the cerebral cortex and thalamus of rats Johnston MV (1998) Selective vulnerability in the neonatal brain. Ann Neu- during development. J Comp Neurol 363:281–295. rol 44:155–156. Towfighi J, Mauger D, Vannucci RC, Vannucci SJ (1997) Influence of age on Johnston MV, Trescher WH, Ishida A, Nakajima W (2001) Neurobiology of the cerebral lesions in an immature rat model of cerebral hypoxia-ischemia: a hypoxic-ischemic injury in the developing brain. Pediatr Res 49:735–741. light microscopic study. Brain Res Dev Brain Res 100:149 –160. Kostovic I, Rakic P (1990) Developmental history of the transient subplate Vannucci RC (1990) Experimental biology of cerebral hypoxia-ischemia: zone in the visual and somatosensory cortex of the macaque monkey and relation to perinatal brain damage. Pediatr Res 27:317–326. human brain. J Comp Neurol 297:441– 470. Vitalis T, Cases O, Callebert J, Launay JM, Price DJ, Seif I, Gaspar P (1998) Kostovic I, Judas M, Rados M, Hrabac P (2002) Laminar organization of the Effects of monoamine oxidase A inhibition on barrel formation in the human fetal cerebrum revealed by histochemical markers and magnetic mouse somatosensory cortex: determination of a sensitive developmental resonance imaging. Cereb Cortex 12:536 –544. period. J Comp Neurol 393:169 –184. Lanzi G, Fazzi E, Uggetti C, Cavallini A, Danova S, Egitto MG, Ginevra OF, Volpe JJ (2001a) Neurobiology of periventricular leukomalacia in the pre- Salati R, Bianchi PE (1998) Cerebral visual impairment in periventricu- mature infant. Pediatr Res 50:553–562. lar leukomalacia. Neuropediatrics 29:145–150. Volpe JJ (2001b) Neurology of the newborn, Ed 4. Philadelphia: Saunders. Lein ES, Finney EM, McQuillen PS, Shatz CJ (1999) Subplate neuron abla- Wood NS, Marlow N, Costeloe K, Gibson AT, Wilkinson AR (2000) Neu- tion alters neurotrophin expression and ocular dominance column for- rologic and developmental disability after extremely preterm birth. EPI- mation. Proc Natl Acad Sci USA 96:13491–13495. Cure Study Group. N Engl J Med 343:378 –384.

Journal

Journal of Neuroscience – Unpaywall

Published: Apr 15, 2003

There are no references for this article.