- Email: [email protected]
Characteristic neuropathology of leukomalacia in extremely low birth weight infants
ELSEVIER
es
Extremely low birth weight periventricular leuko neuropathological and immun Thirteen ELBW infants of 85 infants with at 23 to 27 weeks of gestation, showed a e of distribution e white matter. I ted glial fibrillary astrocytes to be incre g to the intermediate white matter, in all Tumor necrosis factor-a (TNF-@post
lsevier Science
an important problem [3,4]. The brains of ELBW infants are very immature, with a few primary sulci and large subependymal matrix, and often exhibit subependymal and intraventricular hemorrhage. However, few previous studies of periventricular leukomalacia (PVL) in ELBW infants have been performed, and there are many problems to be solved. Tumor necrosis factor-o (TNF-(x) is a polypeptide that can mediate a wide variety of biologic processes, including the growth, development, modulation, and immune response of the brain 151. TNF-cx has been found in the cerebrospinal fluid of newborn infants with meningitis [6] and of newborns with sepsis [7]. All these conditions are associated with an increased risk for PVL. In addition, TNF-cx is increased in microglia in white matter with PVL lesions [8]. P-Amyloid precursor protein (PAPP) is a membranespanning glycoprotcin and has been reported to undergo fast axonal transport to axons and terminals [9]. Recent studies have shown that pAPP accumulation occurs in damaged axons, which disturbs fast axonal transport, and can even occur in an early stage of white matter lesions
w-% Deguchi K, Oguchi K, Takashima S. Characteristic neuropathology of leukomalacia in extremely low birth weight infants. Pediatr Neurol 1997; 16:296-300.
In the present study, we examined white matter damage to elucidate the pathogenesis of PVL in ELBW infants by neuropathologic and immunohistochemical methods, using anti-glial fibrillary acidic protein (GFAP), TNF-a, and pAPP antibodies as markers for reactive astrocytes, activated microglia, and axonal damage, respectively.
Improved obstetric and neonatal intervention has lowered the limit of viability and is increasing the number of surviving extremely low birth weight (ELBW) infants [ 1,2]. However, brain damage in ELRW Infants rem&ins
Thirteen ELBW from postmortem
From the ‘fiDepartment of Mental Retardation and Birth Defect Research: National Institute of Neuroscience; National Center of Neuro!ogy and Psychiatry; Kodaira. Tokyo; and the ‘Department of Pediatrics. School of Medicine, Kitasato University Sagamihara, Kanagawa, Japan.
Communications should be addressed to: Dr. Deguchi; Department of Mental Retardation and Birth Defect Research: National Institute of Neuroscience; NCNP; 4-l - I Ggawahigashi; Kodaira; Tokyo 157, Japan. Received September 13, 1996; Accepted January 29. 1997.
296
PEDIATRIC
NEUROLOGY
Vol.
16 No. 4
atients and
S
infants were selected by neuropathologic examination files of 85 infants with
examination PVL which
0 1997 by Elsevier Science Inc. All rights reserved. PI1 SO887-8994(97)0004 l-6 o 0887-8994/97/$17.00
Table 1. Immumoreactive expression of APP, TNF-OL, and @FAP in EVIL
PVL
85 (1 I/13) 0 (O/9) C.02
Controls P-Value * Number
of cases showing
69 (9113) 0 (O/9) c.05 more
than
62 (8/13) 0 (O/9) c.05
+ + +.
Abbreviations: pAPP = Amyloid precursor protein GFAP = Glial fibrillary acidic protein P-Value = Chi-square value PVL = PSiiVCiItrlCUlX leukomalacia TNF-a = Tumor necrosis factor-a
excluded cases of massive intracranial hemorrhages and anomalies. The infants born at 23 to 27 weeks of gestation (GW), who died at 2 days to 5 months of age after birth, weighed 488 to 955 g at birth, wene divided into three groups. i.e. focal (F), widespread (W), and diffuse (D), according to the distribution of tissue necrosis [ I I]. Nine infants born at 21 to 28 GW, who died at 0 to 2 months of age, and who weighed less than 1,000 g at birth without leukomalacia, intracranial hemorrhages, and anomalies, were used as controls. Neuropathologic examination was performed on large corona1 specimens of the cerebral hemispaeres, including the frontal. parietal and occipital lobes, which were stained with hematoxylin-eosin luxol fast blue. Postmortem examination was performed with of death. lmmunoperoxidase techniques were performed with antibodies against GFAP, TNF-a, and PAPP, on sections 4-pm thick of formalin-fixed and paraffin-embedded tissues. The sections were deparaftinized in xylene and rehydrated in ethanol. Microwave irradiation was performed to retrieve the antigen after endogenous peroxidase activity had been blocked with 0.3% H,O, in methanol. Anti-GFAP, anti-human TNF-a monoclonal (Endogen, I : 100). and anti-PAPP (Boehringer, I : 10) antibodies were used as the primary antibodies. The section\ were incubated for 2 hours at room temperature or at 4°C overnight and then sequentially incubated with biotinylated rabbit anti-mouse immunoglobulin for I hour and peroxidase-conjugated streptavidin for 30 min (both from Nichirei ). The peroxidase activity was vi\ualir.eJ with 0.02% 3.3’-cilanlino21en/en~ 7.4, for 5 min. (DAB. Dojin) and 0.006% H,G in 50 mM Tris
The specificity of the staining was confirmed by negative control experiments, in which normal rabbit serum was applied instead of the primary antibodies. The degree of immunostaining was evaluated as the number of axons positive for pAPP classified into three grades and the number of positive glia per high-magnification view for TNF-(v: and GFAP classified into five grades, respectively: PAPP, -, no staining of axons; +, staining of a small number of axons; and + f. staining of the majority of axons. TNF 01 and GFAP. -, O/625 X IO’ pm’; -I-. 1 to 5: ++, 6 to 10; +++. 11 to 15; and + + + +, more than 15. Statistical analysis with chi-square test was performed on immunoreactive expression of APP. TNF-ol, and 6FAP in PVL (Table 1).
All 13 ELBW infants with PVL showed a widespread distribution of leukomalacia from the dee intermediate white matter. The pathologic features on staining are summarized in Table 2. Spongy changes, cell necrosis, microglial activation, and proliferation of reactive astrocytes and foam cells in and around the areas of leukomalacia were evident in most of the 13 cases, and neovascularization with or wi ut cavity formation was evident in 5 of the 13 cases. wever, it was difficult to detect axonal swellings and coagulation necrosis in these immature brains. A 2-day-old case (Case 11) manifested a gliosis, neovascularization, and cavity formations and was ieved to have prenatal onset leukomalacia. The incide unoreactive expression of VL is shown in Tables 1 TNF-cx, and -positive cells often had a s or various-sized processes surrounding their nuclei. They were increased around necrotic foci in the deep white matter, spreading to the intermediate white matter i 13 cases (62%) with g 1) and markedly itive cells were ablacia. In contrast. sent in the white matter of a TNF-a- itive cells were fou acia born after 23 with leuko (0%) controls. The expression of weig
Table 2.
Case
Gestation (wk)
Necrosis
Swellings
23 23 ‘3 23 24 25 25 2s 26 26 27 27 27
-
--.
9
4 5
6 8 9 IO II I2 I3 Symbols:
-.
none;
35 20
27 53 7 28 12 77 -I6 3
47 5 mo +, slight:
-
-
~~icrQ~tiat Activation
+
Fsa Gel ++
+ -
-t +-
+
+
+
+
+
+
+ +
+
-+ + -
-
2 -
+ -
-
+
+ I
+
+
+
+ +
+
+ -
+
+
t
-
+
-
+. marked
Deguchi
et al: PVL
in EEBW
Infants
297
Table 3. Expression of PAPP, TNF-(u, and GFA leukomnlacia of extremely low birth weight infants
Case
Gestation (wk)
Postnatal Day
pAPP
TNF-a
I
23
3 3 -I 5 6 7 8 9 IO II 12 I3
23 13 24 24 25 25 25 26 26 27 27 27
7 3.5 20 27 54 7 28 12 22 16 2 47 5 mo
+ + ++ ++ ++ -t- + ++ ++ ++ ++ ++
++ ++ + + ++ ++ ++ ++ +
GFAP + +++ ++++ ++ ++ -!- f + + ++++ -I++++ ++++ +++ ++-t+
Abbreviations: pAPP = Amyloid protein GFAP = Glial fibrillary acidic protein TNF-a = Tumor necrosis factor-a precUr.itJl
aavc been many reports OSintraventricular hcmorrhage and developmental sequelae in ELBW infants, but few of PVL in ELBW infants. A retrospective study of 142
infants born at 22 to 25 GW demonstrated that the more immature the infants, the higher the incidence of neonatal complications such as the presence of periventricular or intraventricular hemorrhage or PVL, the incidence of which was 33% at 23 GW, 21% at 24 CW, and 3% at 25 GW [3]. There are several distribution types of PVL lesions. We pre\,;ously classified neonatal leukomalacia into four types; focal, widespread, diffuse, and multicystic encephalomalacia, according to the histologic extent of the necrosis ] 121. In ELBW infants, PVL comprises widespread types. which may be caused by the predisposition of the immature white matter. n the cerebral hemispheres, the perforating branches from the leptomeningeal arteries supply the cortex to the deep white matter and provide an arterial endzone in the periventricular white matter. They are poorly branched, and the ventriculofugal arteries from the lateral striatal and choroidal arteries are also poorly
Figs rtj I. ill tllt~ tkp
Figwt~ rmtter
microglial cytoplasm in and around necrotic foci in the deep white matter spreading to the intermediate white matter (Fig 2). There was no TNF-(x reactivity in glial cells or neurons in the cerebral cortex. BAPP reactivity was found in axons in the cerebral white matter in 11 of 13 infants (85%) with leukomalacia but in none of the 9 controls (0%). The expression of BAPP was noted in axonal swellings, predominantly at the periphery of necrotic foci in the deep to intermediate white matter (Fig 3). BAPP immunoreactivity was marked in swollen axons, but moderate to weak in slightly hypertrophic axons, which were difficult to detect on H-E staining. iscussion
29X
Glitrl.fihrilltrr~ n*ltiW rutrlter
PEDIATRIC
trcitlic proteirl-poJitir,tl trstroglitd of tuw 6. (Origilltll tmg+ljry~~ior~
NEUROLOGY
Vol.
16 No. 4
prolift~rtrtion X 100,)
2. Twwr nrcrosis r!f twse 3. (Origiml
.fut.tor-ol-l~ositi~,~ td1.s rmpiifictrtiorl X200.)
itI tlw
&cl?
white
developed in ELBW infants. On the other hand, deep venous drainage of the cerebral white matter into the internal cerebral veins is relatively well developed [ 131. These characteristics of the vascular architecture may be closely related to the pathogenesis of widespread PVL in ELBW infants. Glial cells are undifferentiated in the cerebral white matter of early fetal brains. ut the neuroglial population increases in areas undergoi myelination during the late fetal period. In controls, GFAP-positive glial cells are scarce in the white matter of ELBW infants and increased in the deep white matter from 28 GW [ 14,151. However, -positive astrocytes were markedly increased around necrotic in the deep to intermediate white matter even in E infants. Several recent studies have shown that astrocytes can produce TNF-(x in response lo viral i lipopolysaccharides (LPS), or cytokineh IS cell cultures are prepared from neonatal mi production of interleukin- I b (IL- I b), TNF human fetal microglia and astrocytes ha after stimulation with LPS, IL-lb. and TNF-CI [ 17,181. TNF-cx has several effects that couid contribute to the development of leukomalacia, such as hypotension. dicseminated intravascular coagulatio 191. and proliferation of astrocytes [ 20,2 11. Our previous study showed that TNF-a immunoreactivity appears in the brain in the third trimester of gestation, and thereafter increases in intensity with advancing age. A double immunofluorescence study demonstrated that TNF-a-immunoreactive cells in neonatal leukomalacia are RCA- l-positive microglial cells 191. In this study of EL W infants. the premature appearance casts was of TNF-cx ~rn~iunoreac~iv~~yin leu~~~~~l~alacia also noted, implying an important role of TN pathogenesis of the white matter lesions sin exerts cytotoxic actions 0 In rat experiments. perikarya (gray matter) binds to the membranes of secretory granules and is then quickly transported along the
axons (white matter) (231. PA immunoreactivity appears 30 minutes after injury to axons adjacent to need1 stabs, and the staining intensity gradually increases wit time. The staining is most intense 15 hours after the injury in neighboring axons, and then gradually weakens in the next 72 hours [24]. In our previous study of term neonates with PVL. pAPP immunoreactiviry appeared in damaged axons 6 hours after birth in the early stage and was detected for the next 22 days [25]. In the present study, pAPP immunoreacaivity in axonal swellings was clearly expressed even in an early stage of necrosis, often in multiple foci, and was widespread in ELBW infants. Therefore, in ELBW infants, cerebral ischemia may induce the multifocal axonal damages and the activation of glial cells. This method is very useful for detecting the necrotic foci in the deep or intermediate white matter in ELBW infants. The expression of pAPP may represent an attempt by neurons to repair their damaged axons in PVL brains. Our neuropathologic and immunohistochemical study demonstrated that axonal damage, marked astrocytosis, and microglial activ occur in the deep to intermediate white matter of EL infants. Therefore, cerebral ischemia may induce the axonal damage, activate microglia, and cause reactive astrocytes to proliferate during PVL formation in ELBW infants. The authors
thank
Prof.
obertsosl
N.
CMT.
Matsuurilti)l
rytichj41jn
his support.
(iJ.
I:tchc4
PC’. kuti
KS. Popu-
liltiOn-hilscd study Of the incidence. complcxiry. and severity of‘ ncuroh)gic di4ilbility among survivor4 ucighing 500 rhrough I250 gram\ ;II ;Iri\on of two birlh c*ohort\. f’CCliiltliC~ 1992:90:750-5. birth: ;I MC,
14
omc
llc‘Ollilti~
J Med
I YO3;329:
]4]
FaZzi
Donahue of infants
E. LNl/i
Chung
lilnil
i)I’ \ i;lbillrj
gc\tntion.
N I+
G, Guard0
A. OnkWo
A. Orcai
S. Pondini
(;.
oukomt’ in vary-low-hirth-w~i~~~~ infant\ with or helll0rrhgc and/or ICUk~M~~ill~Ci~l. Acta Pcdiatr
IY, Benenste
EN. Tumor
necrosis
Induction by lipopolysaccharidc. ol I YYO; 14-I: 2999-3007.
Ih. J In
~acken GH Jr. Mustafa ukin I -bctu ilnd tumor
If-4 fluid in outcome
from
Infect
iv c>f plasma
neonatal
levels
after
production
hy
and intcrleukin-
cnteric
haCilliWy
meningirk
PC-
A. van Ralrn J. et al. Diagnostic
viIlUC
of tumor nwrosk l‘;lcror 01 (TNF cv) and interlcuhin-6 with \cp\i\. Act;1 Pcdlittr l99-I;X3:6Y-1-9. Mir.upuchi M, Tal\a\hima S. Imnlunohi4rochellli~,ll 4. , Bridges
injury
factor-ol
inrerfcron-g
MM. Ramilo 0, Olsen KC’. Ccrcbral necroGs lilCl(W conccnrr3lions. illld
gram-ncgativc
Dis J I989;X: 155-0. ant ESZM, Muntena
r necrosis
axona]
The
I :80X- I I.
atrocytes:
diutr
Al.
31 32 lo 25 weeks’
I SOY-A0 1.
Ncurodevelopm~ntal without perivenrricular I ‘)!I’:8 [5]
PK. Dusinan born
buman
P-amyloid precursor vascular white matter
factor LR.
(Y in neonatal Sivaloganlrthun
head 1rauma
using
protein-immunoreactive lesions. Acta Ncuropathol
Deguchi
leukomala~.;n. S. Early
Pedirltr
dctcclion
01
inlniulio~ytctc8P~~~~istry c,Xr axons in human ccrcbro-
et al: PVL
19c)J;87:55-(1’.
in ELBW
Infants
299
[lo] Suenaga T, Ohnishi K, Nishimura M, Nakamura S. Akiguchi 1, Kimura J. Bundles of amyloid precursor protein-immunoreactive axons in human cerebrovascular white matter lesions. Acta Neuropathol 1994; 87:450-S. [l l] Iida K, Takashima S, Takeuchi Y, Ohno T, Ueda K. Neuropathologic study of newborns with perinatal-onset leukomalacia. Pediatr Neurol 1993;9:45-8. [ 121 Takashima S, Becker LE. Tanaka J. Developmental changes of glial tibrillary acidic protein and myelin bacic protein in perinatal leukomalacia: Relationship to a predisposing factor. Brain Dev 1984;6: 444-50. [13] Kamei A, Houdou M, Mito T, Konomi H, Takashima S. Developmental change in type VI collagen in human cerebral vessels. Pediatr Neural 1992;8: 183-6. [14] Iida K, Takashiama S, Takeuchi Y. Etiologies and distribution of neonatal leukomalacia. Pediatr Neurol 1992;205:205-9. [15] Iida K, Takashima S. Immunohistochemical study on glial cells in brains with periventricular leukomalacia. Neuropathology 1993; 13: 285-90. [la] Merril JE, Koyanagi Y, Zack J. Thomas L, Martin F. Chen ISY. Induction of interleukin- 1 and tumor necrosis factor alpha in brain cultures by human immuno-deficiency virus type I. J Virol 1992;66: 2217-25. [17] Lee SC, Liu W. Dickson DW, Brosnan CF, Berman JW.
300 PEDIATRIC NEUROLOGY
Vol. 16 No. 4
Cytokine production by human fetal microglia and astrocytes. J Immunol 1993;150:2659-67. [18] Chao CC, Hu S, Sheng WS, Peterson PK. Tumor necrosis factor-alpha production by human fetal microglial cells: Regulation by other cytokines. Dev Neurosci 1995; I7:97- 105. [19] Tracy KJ, Beutler B, Lowry SF, et al. Shock and tissue injury induced by recombinant human cachectin. Science 1986;234:470-3. [20] Selmaj KW, Farooq M. Norton WT, Raine CS, Brosnan CF. Proliferation of astrocytes in vitro in response to cytokines. A primary role for tumor necrosis factor. J lmmunol 1990;144: 129-35. [21] Leviton A. Preterm birth and cerebral palsy: Is tumor necrosis factor the missing link? Dev Med Child Neurol 1993;35:549-58. [22] Bruce AJ, Baling W, Kindy MS, et al. Altered neuronal and microglial responses to excitotoxic and ischemic brain injury in mice lacking TNF receptors. Nature Med 1996;2: !88-94. [23] Sisodia SS, Koo EN, Hoffman PN, Perry 6, Price DL. Identification and transport of full-length amyloid precursor proteins in rat peripheral nervous system. J Neurosci 1993;13:3 136-42. [24] Otuka N, Tomonaga M, Ikeda K. Rapid appearance of P-amyloid precursor protein immunoreactivity in damaged axons and reactive glial cells in rat brain following needle stab injury. Brain Res 1991;586: 335-3. [25] Arar Y. Deguchi K, Masashi M, Takashima S. Expression of P-amyloid precursor protein in axons of periventricular leukomalacia bra&. Pediatr Neurol 1995; 13:16 I-3.
es
Extremely low birth weight periventricular leuko neuropathological and immun Thirteen ELBW infants of 85 infants with at 23 to 27 weeks of gestation, showed a e of distribution e white matter. I ted glial fibrillary astrocytes to be incre g to the intermediate white matter, in all Tumor necrosis factor-a (TNF-@post
lsevier Science
an important problem [3,4]. The brains of ELBW infants are very immature, with a few primary sulci and large subependymal matrix, and often exhibit subependymal and intraventricular hemorrhage. However, few previous studies of periventricular leukomalacia (PVL) in ELBW infants have been performed, and there are many problems to be solved. Tumor necrosis factor-o (TNF-(x) is a polypeptide that can mediate a wide variety of biologic processes, including the growth, development, modulation, and immune response of the brain 151. TNF-cx has been found in the cerebrospinal fluid of newborn infants with meningitis [6] and of newborns with sepsis [7]. All these conditions are associated with an increased risk for PVL. In addition, TNF-cx is increased in microglia in white matter with PVL lesions [8]. P-Amyloid precursor protein (PAPP) is a membranespanning glycoprotcin and has been reported to undergo fast axonal transport to axons and terminals [9]. Recent studies have shown that pAPP accumulation occurs in damaged axons, which disturbs fast axonal transport, and can even occur in an early stage of white matter lesions
w-% Deguchi K, Oguchi K, Takashima S. Characteristic neuropathology of leukomalacia in extremely low birth weight infants. Pediatr Neurol 1997; 16:296-300.
In the present study, we examined white matter damage to elucidate the pathogenesis of PVL in ELBW infants by neuropathologic and immunohistochemical methods, using anti-glial fibrillary acidic protein (GFAP), TNF-a, and pAPP antibodies as markers for reactive astrocytes, activated microglia, and axonal damage, respectively.
Improved obstetric and neonatal intervention has lowered the limit of viability and is increasing the number of surviving extremely low birth weight (ELBW) infants [ 1,2]. However, brain damage in ELRW Infants rem&ins
Thirteen ELBW from postmortem
From the ‘fiDepartment of Mental Retardation and Birth Defect Research: National Institute of Neuroscience; National Center of Neuro!ogy and Psychiatry; Kodaira. Tokyo; and the ‘Department of Pediatrics. School of Medicine, Kitasato University Sagamihara, Kanagawa, Japan.
Communications should be addressed to: Dr. Deguchi; Department of Mental Retardation and Birth Defect Research: National Institute of Neuroscience; NCNP; 4-l - I Ggawahigashi; Kodaira; Tokyo 157, Japan. Received September 13, 1996; Accepted January 29. 1997.
296
PEDIATRIC
NEUROLOGY
Vol.
16 No. 4
atients and
S
infants were selected by neuropathologic examination files of 85 infants with
examination PVL which
0 1997 by Elsevier Science Inc. All rights reserved. PI1 SO887-8994(97)0004 l-6 o 0887-8994/97/$17.00
Table 1. Immumoreactive expression of APP, TNF-OL, and @FAP in EVIL
PVL
85 (1 I/13) 0 (O/9) C.02
Controls P-Value * Number
of cases showing
69 (9113) 0 (O/9) c.05 more
than
62 (8/13) 0 (O/9) c.05
+ + +.
Abbreviations: pAPP = Amyloid precursor protein GFAP = Glial fibrillary acidic protein P-Value = Chi-square value PVL = PSiiVCiItrlCUlX leukomalacia TNF-a = Tumor necrosis factor-a
excluded cases of massive intracranial hemorrhages and anomalies. The infants born at 23 to 27 weeks of gestation (GW), who died at 2 days to 5 months of age after birth, weighed 488 to 955 g at birth, wene divided into three groups. i.e. focal (F), widespread (W), and diffuse (D), according to the distribution of tissue necrosis [ I I]. Nine infants born at 21 to 28 GW, who died at 0 to 2 months of age, and who weighed less than 1,000 g at birth without leukomalacia, intracranial hemorrhages, and anomalies, were used as controls. Neuropathologic examination was performed on large corona1 specimens of the cerebral hemispaeres, including the frontal. parietal and occipital lobes, which were stained with hematoxylin-eosin luxol fast blue. Postmortem examination was performed with of death. lmmunoperoxidase techniques were performed with antibodies against GFAP, TNF-a, and PAPP, on sections 4-pm thick of formalin-fixed and paraffin-embedded tissues. The sections were deparaftinized in xylene and rehydrated in ethanol. Microwave irradiation was performed to retrieve the antigen after endogenous peroxidase activity had been blocked with 0.3% H,O, in methanol. Anti-GFAP, anti-human TNF-a monoclonal (Endogen, I : 100). and anti-PAPP (Boehringer, I : 10) antibodies were used as the primary antibodies. The section\ were incubated for 2 hours at room temperature or at 4°C overnight and then sequentially incubated with biotinylated rabbit anti-mouse immunoglobulin for I hour and peroxidase-conjugated streptavidin for 30 min (both from Nichirei ). The peroxidase activity was vi\ualir.eJ with 0.02% 3.3’-cilanlino21en/en~ 7.4, for 5 min. (DAB. Dojin) and 0.006% H,G in 50 mM Tris
The specificity of the staining was confirmed by negative control experiments, in which normal rabbit serum was applied instead of the primary antibodies. The degree of immunostaining was evaluated as the number of axons positive for pAPP classified into three grades and the number of positive glia per high-magnification view for TNF-(v: and GFAP classified into five grades, respectively: PAPP, -, no staining of axons; +, staining of a small number of axons; and + f. staining of the majority of axons. TNF 01 and GFAP. -, O/625 X IO’ pm’; -I-. 1 to 5: ++, 6 to 10; +++. 11 to 15; and + + + +, more than 15. Statistical analysis with chi-square test was performed on immunoreactive expression of APP. TNF-ol, and 6FAP in PVL (Table 1).
All 13 ELBW infants with PVL showed a widespread distribution of leukomalacia from the dee intermediate white matter. The pathologic features on staining are summarized in Table 2. Spongy changes, cell necrosis, microglial activation, and proliferation of reactive astrocytes and foam cells in and around the areas of leukomalacia were evident in most of the 13 cases, and neovascularization with or wi ut cavity formation was evident in 5 of the 13 cases. wever, it was difficult to detect axonal swellings and coagulation necrosis in these immature brains. A 2-day-old case (Case 11) manifested a gliosis, neovascularization, and cavity formations and was ieved to have prenatal onset leukomalacia. The incide unoreactive expression of VL is shown in Tables 1 TNF-cx, and -positive cells often had a s or various-sized processes surrounding their nuclei. They were increased around necrotic foci in the deep white matter, spreading to the intermediate white matter i 13 cases (62%) with g 1) and markedly itive cells were ablacia. In contrast. sent in the white matter of a TNF-a- itive cells were fou acia born after 23 with leuko (0%) controls. The expression of weig
Table 2.
Case
Gestation (wk)
Necrosis
Swellings
23 23 ‘3 23 24 25 25 2s 26 26 27 27 27
-
--.
9
4 5
6 8 9 IO II I2 I3 Symbols:
-.
none;
35 20
27 53 7 28 12 77 -I6 3
47 5 mo +, slight:
-
-
~~icrQ~tiat Activation
+
Fsa Gel ++
+ -
-t +-
+
+
+
+
+
+
+ +
+
-+ + -
-
2 -
+ -
-
+
+ I
+
+
+
+ +
+
+ -
+
+
t
-
+
-
+. marked
Deguchi
et al: PVL
in EEBW
Infants
297
Table 3. Expression of PAPP, TNF-(u, and GFA leukomnlacia of extremely low birth weight infants
Case
Gestation (wk)
Postnatal Day
pAPP
TNF-a
I
23
3 3 -I 5 6 7 8 9 IO II 12 I3
23 13 24 24 25 25 25 26 26 27 27 27
7 3.5 20 27 54 7 28 12 22 16 2 47 5 mo
+ + ++ ++ ++ -t- + ++ ++ ++ ++ ++
++ ++ + + ++ ++ ++ ++ +
GFAP + +++ ++++ ++ ++ -!- f + + ++++ -I++++ ++++ +++ ++-t+
Abbreviations: pAPP = Amyloid protein GFAP = Glial fibrillary acidic protein TNF-a = Tumor necrosis factor-a precUr.itJl
aavc been many reports OSintraventricular hcmorrhage and developmental sequelae in ELBW infants, but few of PVL in ELBW infants. A retrospective study of 142
infants born at 22 to 25 GW demonstrated that the more immature the infants, the higher the incidence of neonatal complications such as the presence of periventricular or intraventricular hemorrhage or PVL, the incidence of which was 33% at 23 GW, 21% at 24 CW, and 3% at 25 GW [3]. There are several distribution types of PVL lesions. We pre\,;ously classified neonatal leukomalacia into four types; focal, widespread, diffuse, and multicystic encephalomalacia, according to the histologic extent of the necrosis ] 121. In ELBW infants, PVL comprises widespread types. which may be caused by the predisposition of the immature white matter. n the cerebral hemispheres, the perforating branches from the leptomeningeal arteries supply the cortex to the deep white matter and provide an arterial endzone in the periventricular white matter. They are poorly branched, and the ventriculofugal arteries from the lateral striatal and choroidal arteries are also poorly
Figs rtj I. ill tllt~ tkp
Figwt~ rmtter
microglial cytoplasm in and around necrotic foci in the deep white matter spreading to the intermediate white matter (Fig 2). There was no TNF-(x reactivity in glial cells or neurons in the cerebral cortex. BAPP reactivity was found in axons in the cerebral white matter in 11 of 13 infants (85%) with leukomalacia but in none of the 9 controls (0%). The expression of BAPP was noted in axonal swellings, predominantly at the periphery of necrotic foci in the deep to intermediate white matter (Fig 3). BAPP immunoreactivity was marked in swollen axons, but moderate to weak in slightly hypertrophic axons, which were difficult to detect on H-E staining. iscussion
29X
Glitrl.fihrilltrr~ n*ltiW rutrlter
PEDIATRIC
trcitlic proteirl-poJitir,tl trstroglitd of tuw 6. (Origilltll tmg+ljry~~ior~
NEUROLOGY
Vol.
16 No. 4
prolift~rtrtion X 100,)
2. Twwr nrcrosis r!f twse 3. (Origiml
.fut.tor-ol-l~ositi~,~ td1.s rmpiifictrtiorl X200.)
itI tlw
&cl?
white
developed in ELBW infants. On the other hand, deep venous drainage of the cerebral white matter into the internal cerebral veins is relatively well developed [ 131. These characteristics of the vascular architecture may be closely related to the pathogenesis of widespread PVL in ELBW infants. Glial cells are undifferentiated in the cerebral white matter of early fetal brains. ut the neuroglial population increases in areas undergoi myelination during the late fetal period. In controls, GFAP-positive glial cells are scarce in the white matter of ELBW infants and increased in the deep white matter from 28 GW [ 14,151. However, -positive astrocytes were markedly increased around necrotic in the deep to intermediate white matter even in E infants. Several recent studies have shown that astrocytes can produce TNF-(x in response lo viral i lipopolysaccharides (LPS), or cytokineh IS cell cultures are prepared from neonatal mi production of interleukin- I b (IL- I b), TNF human fetal microglia and astrocytes ha after stimulation with LPS, IL-lb. and TNF-CI [ 17,181. TNF-cx has several effects that couid contribute to the development of leukomalacia, such as hypotension. dicseminated intravascular coagulatio 191. and proliferation of astrocytes [ 20,2 11. Our previous study showed that TNF-a immunoreactivity appears in the brain in the third trimester of gestation, and thereafter increases in intensity with advancing age. A double immunofluorescence study demonstrated that TNF-a-immunoreactive cells in neonatal leukomalacia are RCA- l-positive microglial cells 191. In this study of EL W infants. the premature appearance casts was of TNF-cx ~rn~iunoreac~iv~~yin leu~~~~~l~alacia also noted, implying an important role of TN pathogenesis of the white matter lesions sin exerts cytotoxic actions 0 In rat experiments. perikarya (gray matter) binds to the membranes of secretory granules and is then quickly transported along the
axons (white matter) (231. PA immunoreactivity appears 30 minutes after injury to axons adjacent to need1 stabs, and the staining intensity gradually increases wit time. The staining is most intense 15 hours after the injury in neighboring axons, and then gradually weakens in the next 72 hours [24]. In our previous study of term neonates with PVL. pAPP immunoreactiviry appeared in damaged axons 6 hours after birth in the early stage and was detected for the next 22 days [25]. In the present study, pAPP immunoreacaivity in axonal swellings was clearly expressed even in an early stage of necrosis, often in multiple foci, and was widespread in ELBW infants. Therefore, in ELBW infants, cerebral ischemia may induce the multifocal axonal damages and the activation of glial cells. This method is very useful for detecting the necrotic foci in the deep or intermediate white matter in ELBW infants. The expression of pAPP may represent an attempt by neurons to repair their damaged axons in PVL brains. Our neuropathologic and immunohistochemical study demonstrated that axonal damage, marked astrocytosis, and microglial activ occur in the deep to intermediate white matter of EL infants. Therefore, cerebral ischemia may induce the axonal damage, activate microglia, and cause reactive astrocytes to proliferate during PVL formation in ELBW infants. The authors
thank
Prof.
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N.
CMT.
Matsuurilti)l
rytichj41jn
his support.
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KS. Popu-
liltiOn-hilscd study Of the incidence. complcxiry. and severity of‘ ncuroh)gic di4ilbility among survivor4 ucighing 500 rhrough I250 gram\ ;II ;Iri\on of two birlh c*ohort\. f’CCliiltliC~ 1992:90:750-5. birth: ;I MC,
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IY, Benenste
EN. Tumor
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~acken GH Jr. Mustafa ukin I -bctu ilnd tumor
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of tumor nwrosk l‘;lcror 01 (TNF cv) and interlcuhin-6 with \cp\i\. Act;1 Pcdlittr l99-I;X3:6Y-1-9. Mir.upuchi M, Tal\a\hima S. Imnlunohi4rochellli~,ll 4. , Bridges
injury
factor-ol
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MM. Ramilo 0, Olsen KC’. Ccrcbral necroGs lilCl(W conccnrr3lions. illld
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PK. Dusinan born
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factor LR.
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head 1rauma
using
protein-immunoreactive lesions. Acta Ncuropathol
Deguchi
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et al: PVL
19c)J;87:55-(1’.
in ELBW
Infants
299
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