Introduction 41 1 4 8 39 42 13 4 1 8 23 24 5 25 35 17 18 17 18 19 34 n n n Materials and methods Case selection criteria The neuropathology of all premature infants (<37 gestational weeks at birth) autopsied between 1997–1999 at Children’s Hospital Boston was retrospectively reviewed. This time-frame was selected because it represents a modern era of intensive care management of premature infants in a Level 3 neonatal intensive care nursery. It also represents a period at our hospital when the brain and spinal cord were extensively sampled in a relatively standardized fashion, whether or not macroscopic lesions were apparent, thereby permitting a systematic neuropathologic survey in a large dataset. Parental authorization of the use of autopsy human tissue for research was given in each case. Microscopic slide review A median of 15 (range 9–20) hematoxylin–eosin (H&E) or H&E/Luxol-fast-blue stained sections was examined from each case. These sections included cerebral cortex from all lobes, thalamus (at the level of lateral geniculate nucleus and including the dorsomedial and lateral posterior nuclei), hypothalamus, caudate, putamen, globus pallidus, hippocampus (level of lateral geniculate nucleus), amygdala, cerebellar dentate nucleus, cerebellar cortex (including Purkinje cells and granule cell layer), midbrain, pons, and medulla. 23 2 2 2 24 24 Glial fibrillary acidic protein (GFAP) immunohistochemistry Four-micron thick formalin-fixed paraffin-embedded sections of frontal cortex could be cut from 27 of the 41 cases and were immunostained with mouse anti-GFAP antibody (1:500, #SM1-22R, Covance, Berkeley, CA). Negative controls were performed without primary antibody. Scoring of GFAP stained sections was performed by counting positive cells/hpf, in the most intensely immunopositive region of frontal cortex after a survey of all fields. Reactive astrocytes were defined as those cells with substantial cytoplasmic GFAP staining around a nucleus. Other non-reactive cortical astrocytes with limited cytoplasmic staining had GFAP positive processes that were generally perpendicular to the glial limitans were counted separately. The grading system was: 0, no staining; 1, 1–10 cells/hpf; 2, 11–20 cells/hpf; and 3 > 20 cells/hpf. Two observers (CRP, HCK) scored each case without knowledge of the white matter group. O4 and GFAP double-labeling immunofluorescence 36 Statistical analysis 23 24 2 1 P Table 1 Clinicopathologic variables of the three white matter study groups for comparison of gray matter injuries  Mean ± SD; median, (range) or percent P n n n Three-way PVL versus DWMG Demographics  Gestational age (weeks) 32.8 ± 3.1; 34, (26–36) 31.6 ± 3.8; 33, (24–36) 26.5 ± 2.3; 27, (23–30) 0.003 0.282  Postnatal age (weeks) 3.7 ± 4.1; 2.3, (0.1–15) 3.4 ± 4.0; 1.2, (0.1–12.0) 0.8 ± 1.2; 0.1, (0.1–3.0) 0.141   Postconceptional age (weeks) 36.5 ± 5.4; 35.5, (26–52) 34.8 ± 4.5; 35.0, (28.4–48.0) 27.3 ± 2.8; 28.3, (23.1–30.3) 0.001 0.293  Length of ICU stay (days) 13.8 ± 15.9; 9.5, (0–56)  23.8 ± 34.6; 5.0, (1–96)  5.0 ± 8.9; 1.5, (0.04–23.0)  0.419  Race 8/17; 47% 9/17; 53% 5/7; 71% 0.801   African-American 2/17; 12% 2/17; 12% 1/7; 14%  Unknown 7/17; 41% 6/17; 35% 1/7; 14%  Percent male 9/17, 53% 7/17, 41% 4/7, 57% 0.702  Postmortem interval (h) 16.0 ± 7.7; 18, (2–30) 23.4 ± 19.6; 17, (2–72)  36.3 ± 42.4; 22, (16–132) 0.364  Birth weight (g) 1576 ± 717; 1380, (780–2700) 1832 ± 1048; 2100, (610–3800) 925 ± 408; 930, (440–1400) 0.232  Brain weight (g) 245.0 ± 81.1; 257.5, (110–340) 255.0 ± 72.1; 245, (124–380)  127.6 ± 40.7; 140.0, (63.8–178.0) 0.003 0.905 Body weight (g) 2423.1 ± 1165.0; 2225, (760–4875) 3276.2 ± 4706.0; 2290, (1150–21300) 947.9 ± 359.2; 915, (440–1400) 0.002 0.815 Body length (cm) 42.0 ± 7.8; 41.5, (26–56) 43.2 ± 4.4; 44.0, (36–51) 34.7 ± 4.0; 32.5, (30–41) 0.007 0.589 Twin or other multiple gestation 3/17, 18% 4/17, 24% 2/7, 29% 0.824  a 4/17, 24% 2/17, 12% 3/7, 43% 0.242  Cesarean section 10/17, 59% 12/17, 71% 3/7, 43% 0.436  Cardiorespiratory factors Apgar score at 1 min 4.1 ± 2.4; 4, (1–8)  4.7 ± 2.9; 5, (0–9)  3.5 ± 2.2; 3, (1–7)  0.691  Apgar score at 5 min 5.2 ± 2.9; 6, (1–9)  6.6 ± 2.9; 7, (0–10)  5.0 ± 2.8; 5.5, (1–8)  0.405  Acute respiratory distress syndrome 7/17, 41% 10/17, 59% 5/7, 71% 0.344  Mechanical ventilation 13/17, 77% 13/17, 77% 7/7, 100% 0.359  Length of mechanical ventilation (days) 8.4 ± 11.6; 3.0, (0–38) 3.5 ± 6.3; 1.0, (1–19)  4.4 ± 8.2; 1.0, (0.04–23.0)  0.383  Extracorpeal membrane oxygenation 1/17%, 6% 0/17%, 0% 0/7, 0% 0.485  Continuous positive airway pressure 0/17, 0% 3/17, 18% 1/7, 14% 0.202  Cardiopulmonary resuscitation any time during hospitalization 5/17, 29% 7/17, 41% 5/7, 71% 0.165  a 2/17, 12% 2/17, 12% 1/7, 14% 0.983  Infectious/inflammatory factors a 4/17, 24% 2/17, 12% 0/7, 0% 0.303  Maternal fever at delivery 0/15, 0% 2/9, 22% 0/4, 0% 0.103  Maternal history of urinary tract infection  0/15, 0% 0/10, 0% 1/4, 25% 0.039 NA Chorioamnionitis 1/15, 7% 2/10, 20% 1/5, 20% 0.562  a 1/17, 6% 4/17, 24% 2/7, 29% 0.415  Clinical diagnosis of sepsis 8/17, 47% 8/17, 47% 3/7, 43% 0.980  P P P  NS a Results Clinical and autopsy data Seventeen cases (41%) fulfilled the criteria for PVL, while 17 cases (41%) had DWMG, and there were 7 so-called Negative cases (17%). Pregnancy, labor and delivery 1 1 1 1 Syndromes Twenty-four percent of cases had a constellation of findings classified as a genetic/developmental syndrome, e.g., Treacher-Collins syndrome, Potter’s sequence, and osteogenesis imperfecta in three PVL cases; Down’s syndrome in a DWMG case; and Fryns syndrome, Beckwith-Wiedemann syndrome, and VACTERL association in three Negative cases. Excluding these cases from the analysis had no significant effect on the results for the different acquired lesions analyzed semi-quantitatively, i.e neuronal loss and gliosis (data not shown), and thus, their data were combined with that of the non-syndromic cases in the complete analysis reported below. Postnatal period 1 1 1 1 White matter findings 2 2 2 2 2 P Table 2 Distribution of white matter lesions in 41 autopsied premature infants by anatomic site and postconceptional age (weeks) White matter site Overall incidence Incidence by postconceptional age (weeks)  P 23–29 30–36 37+ Frontal lobe PVL 11/40 (28%) 0/8 (0%) 6/22 (27%) 5/10 (50%) 0.025 DWMG 31/40 (78%) 2/8 (25%) 19/22 (86%) 10/10 (100%) 0.004 Temporal lobe PVL 4/33 (12%) 0/6 (0%) 3/22 (14%) 1/5 (20%) NS DWMG 22/33 (67%) 1/6 (17%) 16/22 (73%) 5/5 (100%) 0.006 Parietal lobe PVL 7/28 (25%) 0/6 (0%) 6/16 (38%) 1/6 (17%) NS DWMG 20/28 (71%) 1/6 (17%) 13/16 (82%) 6/6 (100%) 0.010 Occipital lobe PVL 8/35 (23%) 1/5 (20%) 5/23 (22%) 2/7 (29%) NS DWMG 28/35 (80%) 3/5 (60%) 19/23 (83%) 6/7 (86%) NS Corpus callosum PVL 2/22 (9%) 0/3 (0%) 1/14 (7%) 1/5 (20%) NS DWMG 14/22 (64%) 0/3 (0%) 10/14 (71%) 4/5 (80%) 0.049 Internal capsule PVL 5/27 (19%) 0/2 (0%) 3/18 (17%) 2/7 (29%) NS DWMG 18/27 (67%) 0/2 (0%) 12/18 (67%) 6/7 (86%) NS Cerebellum PVL 3/40 (8%) 0/8 (0%) 2/22 (9%) 1/10 (10%) NS DWMG 34/40 (85%) 4/8 (50%) 21/22 (95%) 9/10 (90%) 0.012 P NS PVL DWMG 1 Fig. 1 a b c  Gray matter lesions associated with PVL in the premature infant Acute neuronal necrosis P P Neuronal loss and gliosis 2 3 3 4 2 3 3 P 4 2 3 3 4 3 4 Fig. 2 a b c d asterisk b  Fig. 3 a b c d Arrows b c  Table 3 Incidence and severity of neuronal loss in PVL, DWMG and Negative cases  Overall incidence Incidence of severity 2–3 PVL DWMG Negative P PVL DWMG Negative P Neuronal loss Cerebral cortex Frontal cortex 13% (2/16) 0% (0/17) 0% (0/7) 0.477 6% (1/16) 0% (0/17) 0% (0/7) 0.575 Temporal cortex 0% (0/15) 0% (0/13) 0% (0/6) 1.000 0% (0/15) 0% (0/13) 0% (0/6) 1.000 Parietal cortex 8% (1/13) 0% (0/11) 0% (0/6) 1.000 8% (1/13) 0% (0/11) 0% (0/6) 1.000 Occipital Cortex 0% (0/15) 0% (0/16) 0% (0/4) 1.000 0% (0/15) 0% (0/16) 0% (0/4) 1.000 Deep gray nuclei Thalamus 38% (6/16) 0% (0/17) 0% (0/7) 0.005 38% (6/16) 0% (0/17) 0% (0/7) 0.005 Hypothalamus 20% (2/10) 0% (0/10) 0% (0/2) 0.567 10% (1/10) 0% (0/10) 0% (0/2) 1.000 Caudate 13% (2/15) 0% (0/16) 0% (0/7) 0.329 13% (2/15) 0% (0/16) 0% (0/7) 0.329 Putamen 13% (2/16) 0% (0/17) 0% (0/7) 0.477 13% (2/16) 0% (0/17) 0% (0/7) 0.477 Globus pallidus 33% (5/15) 0% (0/15) 0% (0/6) 0.028 33% (5/15) 0% (0/15) 0% (0/6) 0.028 Cerebellum and relay nuclei Basis pontis 21% (3/14) 0% (0/14) 0% (0/7) 0.206 14% (2/14) 0% (0/14) 0% (0/7) 0.341 Inferior olive 15% (2/13) 8% (1/13) 20% (1/5) 0.807 8% (1/13) 8% (1/13) 20% (1/5) 0.549 Cerebellar cortex 24% (4/17) 6% (1/16) 14% (1/7) 0.449 24% (4/17) 6% (1/16) 14% (1/7) 0.449 Dentate 29% (4/14) 0% (0/15) 0% (0/6) 0.031 29% (4/14) 0% (0/15) 0% (0/6) 0.031 Limbic structures Hippocampus 33% (5/13) 0% (0/14) 14% (1/7) 0.055 33% (5/15) 0% (0/14) 14% (1/7) 0.055 Amygdala 0% (0/6) 0% (0/3) 0% (0/2) 1.000 0% (0/6) 0% (0/3) 0% (0/2) 1.000 Substantia inominata 29% (2/7) 0% (0/3) 0% (0/1) 1.000 29% (2/7) 0% (0/3) 0% (0/1) 1.000 Brainstem 14% (2/14) 0% (0/14) 0% (0/5) 0.629 14% (2/14) 0% (0/14) 0% (0/5) 0.629 PVL DWMG P Table 4 Incidence and severity of gliosis in PVL, DWMG and Negative cases  Overall incidence Incidence of severity 2–3 PVL DWMG Negative P PVL DWMG Negative P Gliosis Cerebral cortex Frontal cortex 31% (5/16) 6% (1/17) 0% (0/7) 0.102 13% (2/16) 0% (0/17) 0% (0/7) 0.477 Temporal cortex 20% (3/15) 8% (1/13) 0% (0/6) 0.495 0% (0/15) 0% (0/13) 0% (0/6) 1.000 Parietal cortex 23% (3/13) 9% (1/11) 0% (0/6) 0.499 8% (1/13) 9% (1/11) 0% (0/6) 1.000 Occipital cortex 27% (4/15) 0% (0/16) 0% (0/4) 0.054 13% (2/15) 0% (0/16) 0% (0/4) 0.395 Deep gray nuclei Thalamus 56% (9/16) 18% (3/17) 14% (1/7) 0.031 19% (3/16) 0% (0/17) 14% (1/7) 0.161 Hypothalamus 40% (4/10) 10% (1/10) 50% (1/2) 0.264 20% (2/10) 0% (0/10) 50% (1/2) 0.130 Caudate 60% (9/15) 19% (3/16) 14% (1/7) 0.028 13% (2/15) 6% (1/16) 14% (1/7) 0.659 Putamen 50% (8/16) 12% (2/17) 14% (1/7) 0.044 19% (3/16) 0% (0/17) 0% (0/7) 0.130 Globus Pallidus 60% (9/15) 47% (7/15) 0% (0/6) 0.040 20% (3/15) 7% (1/15) 0% (0/6) 0.492 Cerebellum and relay nuclei Basis pontis 100% (14/14) 79% (11/14) 29% (2/7) 0.001 36% (5/14) 21% (3/14) 14% (1/7) 0.684 Inferior olive 92% (12/13) 92% (12/13) 80% (4/5) 0.549 62% (8/13) 54% (7/13) 20% (1/5) 0.400 Cerebellar cortex 29% (5/17) 6% (1/16) 14% (1/7) 0.259 12% (2/17) 6% (1/16) 0% (0/7) 1.000 Dentate 43% (6/14) 13% (2/15) 17% (1/6) 0.177 21% (3/14) 0% (0/15) 0% (0/6) 0.125 Limbic structures Hippocampus 47% (7/15) 7% (1/14) 29% (2/7) 0.056 20% (3/15) 0% (0/14) 29% (2/7) 0.143 Amygdala 50% (3/6) 0% (0/3) 0% (0/2) 0.3273 0% (0/6) 0% (0/3) 0% (0/2) 1.000 Substantia inominata 29% (2/7) 0% (0/3) 0% (0/1) 1.000 0% (0/7) 0% (0/3) 0% (0/1) 1.000 Brainstem 43% (6/14) 20% (3/14) 20% (1/20) 0.518 7% (1/14) 0% (0/14) 0% (0/5) 1.000 PVL P n n n 4 Fig. 4 a b  Discussion 5 Fig. 5 a b right of panel left of panel  24 3 23 40 9 23 9 23 40 27 27 15 7 38 33 7 26 21 37 17 18 19 34 35 10 12 14 22 30 31 16 6 20 28 29 32 21 37 41 In conclusion, this study draws attention to the combination of white and gray matter injury in the brains of preterm infants dying in the perinatal period by the term “perinatal panencephalopathy”. Our findings suggest that future treatment strategies should target both white and gray matter damage to prevent the neurologic deficits in survivors of prematurity.