animals : all experiments were performed using female wistar imamichi rats , 
 910 weeks of age , 218.45  2.09 g ( mean  se ) in weight , and obtained from the institute of 
 animal reproduction ( kasumigaura , japan ) .
the animals were reared in a room with the 
 temperature controlled at 22  2c , humidity at 50  5% , with ventilation 11 times per hr , 
 lighting at 12:12-hr light / dark cycle ( light cycle , 7:0019:00 ) and given standard chow 
 ( ce-2 ; nihon clea , tokyo , japan ) .
the present experiments were performed following the 
 provisions approved by the animal research committee of tottori university . experimental design : a total of 40 animals were divided into 2 groups as 
 follows : ( 1 ) saline - treated control rats ( n=20 ) , ( 2 ) mtx - treated rats ( n=20 ) .
day 0 of gestation ( gd 0 ) was designated 
 as the day when the presence of a vaginal plug was identified .
the rats received 
 intraperitoneal injections ( i.p . ) with mtx ( 30 mg / kg body weight ) or saline ( the control ) on 
 gd 13 .
the specific timing of mtx administration was selected , because the injection of dna 
 damaging chemicals , such as ethylnitrosourea ( enu ) , 6-mercaptopurine ( 6-mp ) , busulfan 
 , t-2 toxin , 1--d - arabinofuranosylcytosine ( ara - c ) , 5-azacytidine ( 5azc ) [ 39 , 40 ] and 5-fluorouracil ( 5-fu ) , on gd13 induced apoptosis in neuroepithelial cells of telencephalon 
 in rat fetal brain . the dose level in the present study
was decided , because this dose was 
 equivalent to 10% of the ld50 in intraperitoneal injection in rats or 1530% of dosage used 
 in high - dose cancer therapy in human [ 12 , 15 , 23 ] .
fetus 
 samples were collected under pentobarbital anesthesia ( 100 mg / kg , i.p . ) 6 , 12 , 24 , 36 and 48 
 hr , respectively , after mtx administration .
they were removed from the uterus and weighed , 
 and their body lengths were measured . histopathological examination : for histopathological examination , all 
 fetuses were fixed in 10% neutral buffered formalin and then embedded in paraffin .
the 
 tissues of telencephalon , diencephalon , mesencephalon and metencephalon in 12 fetal brain 
 samples per group consisted of 3 fetuses ramdomly selected per dam in each group were 
 sectioned at 2 m thickness , stained with hematoxylin and eosin and 
 examined with light microscopy .
the number of pyknotic cells or mitotic cells was counted 
 from over 1,000 neuroepithelial cells in the telencephalon , diencephalon , mesencephalon and 
 metencephalon for each fetus by light microscopy , and the pyknotic index and mitotic index 
 were calculated as the percentage of pyknotic cells or mitotic cells from out of the total 
 number of neuroepithelial cells counted .
tunel method : dna - fragmented neuroepithelial cells in telencephalon were 
 detected by terminal deoxynucleotidyl - transferase ( tdt)-mediated deoxyuridine 
 triphosphate - digoxigenin ( dutp ) nick - end labeling ( tunel ) , which was performed using an 
 in situ apoptosis detection kit ( trevigen , inc . ,
the number of tunel - positive cells was obtained from over 1,000 neuroepithelial 
 cells in the telencephalon for each fetus by light microscopy , and the tunel - index was 
 calculated as the percentage of tunel - positive cells out of the total number of 
 neuroepithelial cells counted .
immunohistochemical examinations of rat fetuses : immunohistochemical 
 staining was performed by a labeled - polymer method using histofine simple stain max - po ( r ) 
 ( nichirei , tokyo , japan ) . to retrieve the antigen , tissue sections for the detection of 
 cleaved caspase-3 antigen
were immersed in citrate buffer , ph 6.0 ( dako , glostrup , denmark ) 
 and autoclaved for 15 min at 121c ; and tissue sections for the detection of phospho - histone 
 h3 antigen were immersed in citrate buffer , ph 6.0 ( dako ) and microwaved for 15 min . 

endogenous peroxidase activity was quenched by immersing the sections in 3% hydrogen 
 peroxide in methanol for 15 min .
the sections were incubated with the cleaved caspase-3 
 rabbit polyclonal antibody ( 1:300 dilution ; cell signaling technology , inc . , danvers , ma , 
 u.s.a . ) at 4c overnight ; and the sections were incubated with the phospho - histone h3 rabbit 
 monoclonal antibody ( 1:1,500 dilution ; abcam , tokyo , japan ) for 30 min at room temperature . 

then , these sections were treated with histofine simple stain max - po ( r ) ( nichirei ) for 30 
 min at room temperature .
they were exposed to a 3,3-diaminobenzidine solution containing 
 hydrogen peroxide ( nichirei ) to facilitate a peroxidase color reaction and then 
 counterstained with mayer s hematoxylin .
the number of cleaved caspase-3- or phospho - histone 
 h3-positive cells was counted from over 1,000 neuroepithelial cells in the telencephalon for 
 each fetus by light microscopy , and the cleaved caspase-3- or phospho - histone h3-index was 
 calculated as the percentage of cleaved caspase-3- or phospho - histone h3-positive cells out 
 of the total number of neuroepithelial cells counted .
statistical analysis : means  standard error ( se ) of the individual litter 
 value was calculated .
effects of mtx on rat fetuses : six , 12 and 24 hr after mtx treatment , 
 there were no significant differences in the number of living fetuses per litter and the 
 fetal mortality rates between the control group and mtx - treated group ( table 1table 1 .
of damstotal no . of live fetusesliving fetuses per litterdead fetus ratio ( % ) 6 hrcontrol45814.59  0.509.03  3.03mtx45714.25  0.633.14 1.8212 hrcontrol45614.00 
0.417.93  3.00mtx46315.75  2.5015.92  10.6724 hrcontrol45814.50  1.447.63  4.81mtx45714.25  0.956.48  4.4436 hrcontrol46215.50  1.193.58  3.58mtx44411.00  0.41 * 30.30  5.41**48 hrcontrol45614.00  1.004.73  3.15mtx410.25  0.2598.53  1.48***values are expressed as means  se .
* , * * , * * * : significantly different from control 
 at p<0.05 , p<0.01 , 
 p<0.001 , respectively ( student s t - test ) .
 : 
 significantly different from control at p<0.001 ( welch s 
 t - test ) . )
the number of living fetuses significantly declined , and fetal mortality 
 rates significantly increased at 36 hr in the mtx - treated group , compared to those of the 
 control group ( table 1 ) .
almost all fetuses died 
 by 48 hr after mtx treatment ( table 1 ) .
* , * * , * * * : significantly different from control 
 at p<0.05 , p<0.01 , 
 p<0.001 , respectively ( student s t - test ) .
 : 
 significantly different from control at p<0.001 ( welch s 
 t - test ) .
histopathological findings of rat fetal brain : in the control group , 
 pyknotic changes in neuroepithelial cells were rarely observed in any layers of the 
 telencephalic wall throughout the experimental period ( figs . 1 and 2fig
. 1.histopathological findings of telencephalic wall of rat fetal brain in the control 
 group 6 hr after treatment ( a ) and the mtx - treated group at 6 ( b ) , 12 ( c ) , 24 ( d ) and 
 36 hr ( e ) , respectively .
2.changes in the pyknotic index ( % ) in telencephalic wall of rat fetal brain .
 ,  : significantly different from control at 
 p<0.05 , p<0.001 , respectively ( welch s 
 t - test ) . )
histopathological findings of telencephalic wall of rat fetal brain in the control 
 group 6 hr after treatment ( a ) and the mtx - treated group at 6 ( b ) , 12 ( c ) , 24 ( d ) and 
 36 hr ( e ) , respectively .
bar=30 m . changes in the pyknotic index ( % ) in telencephalic wall of rat fetal brain .
 ,  : significantly different from control at 
 p<0.05 , p<0.001 , respectively ( welch s 
 t - test ) .
six hr after treatment in the mtx - treated group , pyknotic neuroepithelial cells appeared in 
 the telencephalic wall and scattered throughout that wall ( fig .
then , 1236 hr after mtx treatment , pyknotic 
 neuroepithelial cells drastically increased and were diffusely distributed throughout the 
 telencephalic wall ( figs .
, 
 neuroepithelial cells were eliminated and showed low cell density in the telencephalon of 
 mtx - treated group ( fig .
although mitotic 
 neuroepithelial cells were located along the ventricular layer of the telencephalic wall in 
 the control group , they were rarely observed in the same region at 636 hr in the 
 mtx - treated group ( fig .
1b1e ) . in the telencephalon of the control group , tunel - positive neuroepithelial cells were rarely 
 observed in any layers of the telencephalic wall throughout the experimental period ( figs
3.tunel-positive cells in telencephalic wall of rat fetal brain in the control group 6 
 hr after treatment ( a ) and the mtx - treated group at 6 ( b ) , 12 ( c ) , 24 ( d ) and 36 hr 
 ( e ) .
most of the pyknotic neuroepithelial cells were positively stained by the tunel 
 method at 12 , 24 and 36 hr in the mtx - treated group ( fig . 

3c3e ) , while there were few pyknotic neuroepithelial cells positively stained by 
 the tunel method at 6 hr ( fig .
the index of tunel - positive neuroepithelial cells in the telencephalic 
 wall significantly increased at 12 and 24 hr and peaked at 36 hr ( fig .
4.changes in the tunel index ( % ) in telencephalic wall of rat fetal brain .
 ,  : significantly different from control at 
 p<0.01 , p<0.001 , respectively ( welch s 
 t - test ) . ) .
tunel - positive cells in telencephalic wall of rat fetal brain in the control group 6 
 hr after treatment ( a ) and the mtx - treated group at 6 ( b ) , 12 ( c ) , 24 ( d ) and 36 hr 
 ( e ) .
bar=30 m . changes in the tunel index ( % ) in telencephalic wall of rat fetal brain .
 ,  : significantly different from control at 
 p<0.01 , p<0.001 , respectively ( welch s 
 t - test )
. cleaved caspase-3-positive neuroepithelial cells were rarely observed in the telencephalon 
 of control group throughout the experimental period ( figs . 5a and 6fig .
5.immunohistochemical expression of cleaved caspase-3 in telencephalic wall of rat 
 fetal brain in the control group 6 hr after treatment ( a ) and the mtx - treated group at 
 6 ( b ) , 12 ( c ) , 24 ( d ) and 36 hr ( e ) .
bar=30 m ) , although in the mtx - treated group , almost all pyknotic neuroepithelial cells were 
 immunohistochemically positive for cleaved caspase-3 throughout the experimental period 
 ( fig .
5b5e ) . in the mtx - treated group , the 
 index of cleaved caspase-3-positive neuroepithelial cells in the telencephalon significantly 
 increased at 6 , 12 and 24 hr and peaked at 36 hr ( fig . 

 ,  : significantly 
 different from control at p<0.05 , p<0.001 , 
 respectively ( welch s t - test ) . )
immunohistochemical expression of cleaved caspase-3 in telencephalic wall of rat 
 fetal brain in the control group 6 hr after treatment ( a ) and the mtx - treated group at 
 6 ( b ) , 12 ( c ) , 24 ( d ) and 36 hr ( e ) . bar=30 m cleaved caspase-3 index .
 ,  : significantly 
 different from control at p<0.05 , p<0.001 , 
 respectively ( welch s t - test ) .
phospho - histone h3-positive neuroepithelial cells were located along the ventricular layer 
 of the telencephalic wall in the control group , while there were fewer phospho - histone 
 h3-positive cells at 636 hr in the same region of the mtx - treated group than in the control 
 group ( fig .
7.immunohistochemical expression of phospho - histone h3 in telencephalic wall of rat 
 fetal brain in the control group 6 hr after treatment ( a ) and the mtx - treated group at 
 6 ( b ) , 12 ( c ) , 24 ( d ) and 36 hr ( e ) , respectively .
the index of phospho - histone h3-positive neuroepithelial cells in telencephalon 
 decreased significantly at 6 hr compared with the control group and maintained that low 
 level throughout the experimental period ( fig . 

values are expressed as means  se . * * * : significantly 
 different from control at p<0.001 ( student s 
 t - test ) .  : significantly different from control at 
 p<0.01 ( welch s t - test ) . ) .
immunohistochemical expression of phospho - histone h3 in telencephalic wall of rat 
 fetal brain in the control group 6 hr after treatment ( a ) and the mtx - treated group at 
 6 ( b ) , 12 ( c ) , 24 ( d ) and 36 hr ( e ) , respectively .
values are expressed as means  se . * * * : significantly 
 different from control at p<0.001 ( student s 
 t - test ) .  : significantly different from control at 
 p<0.01 ( welch s t - test ) . in the mtx - treated group , pyknotic indices of telencephalon and diencephalon
significantly 
 increased at 1236 hr , and those of the mesencephalon significantly increased at 24 and 36 
 hr , compared to the control group ( table 
 2table 2.pyknotic index ( % ) in rat fetal brain treated by mtx6 hr12 hr24 hr36 hrtelencephaloncontrol0.03  0.030.05  0.050.10  0.070.28  0.14mtx2.68  0.8519.98  2.4534.82  2.1743.33  3.07diencephaloncontrol0.61  0.280.79  0.300.66  0.360.81  0.30mtx1.61  0.3814.31  1.5320.88  2.4420.65  1.67mesencephaloncontrol0.39  0.040.41  0.110.29  0.110.47  0.12mtx0.37  0.187.78  2.8826.90  5.4536.56  3.99metencephaloncontrol0.00  0.000.00 
0.000.00  0.000.07  0.05mtx0.04  0.020.00  0.002.34  1.523.88  0.72values are expressed as means  se ( % ) .
 ,  ,  : significantly different from 
 control at p<0.05 , p<0.01 , 
 p<0.001 respectively ( welch s t - test ) . )
the pyknotic index of metencephalon significantly increased at 36 hr 
 compared to control group , and it showed a clearly lower value , compared to those of the 
 telencephalon , diencephalon and mesencephalon ( table 
 2 ) . in the mtx - treated group , there were fewer pyknotic neuroepithelial cells in 
 the metencephalon than in the telencephalon , diencephalon and mesencephalon . in the 
 mtx - treated group , while mitotic indices of telencephalon , diencephalon and mesencephalon 
 decreased significantly at 636 hr , that of metencephalon decreased significantly at 6 and 
 12 hr compared to the control group ( table 
 3table 3 .
mitotic index ( % ) in rat fetal brain treated by mtx6 hr12 hr24 hr36 hrtelencephaloncontrol4.56  0.204.48  0.254.82  0.244.50  0.09mtx0.53  0.050.24  0.030.16  0.080.22  0.13diencephaloncontrol3.01 
0.283.30  0.332.56  0.363.66  0.28mtx0.37  0.080.57  0.090.37  0.090.40  0.20mesencephaloncontrol3.64  0.433.76  0.353.51  0.444.53  0.34mtx0.64  0.170.96  0.100.22  0.071.13  0.27metencephaloncontrol3.12  0.643.25  0.251.37  0.251.25  0.10mtx0.67  0.081.40  0.351.53  0.471.60  0.26values are expressed as means  se ( % ) . * * , * * * : significantly different from control 
 at p<0.01 , p<0.001 respectively ( student s 
 t - test ) .
 ,  ,  : significantly different from control at 
 p<0.05 , p<0.01 , p<0.001 
 respectively ( welch s t - test ) . ) .
 ,  ,  : significantly different from 
 control at p<0.05 , p<0.01 , 
 p<0.001 respectively ( welch s t - test ) .
values are expressed as means  se ( % ) . * * , * * * : significantly different from control 
 at p<0.01 , p<0.001 respectively ( student s 
 t - test ) .
 ,  ,  : significantly different from control at 
 p<0.05 , p<0.01 , p<0.001 
 respectively ( welch s t - test ) .
it has been reported that microcephaly can occur as one of the central nervous system 
 anomalies of mtx embryopathy in humans [ 1 , 8 ] .
the pathogenesis of microcephaly was closely 
 associated with excessive neuronal death [ 7 , 31 , 38 ] .
the cause 
 of mtx - induced microcephaly may be also associated with excessive neuronal death . in the 
 present study ,
the majority of pyknotic neuroepithelial cells in the telencephalic wall were 
 positive for tunel staining and cleaved caspase-3 .
cleavage of caspase-3 is known to be 
 involved in cell apoptosis and is recognized as an apoptosis marker .
these results indicate that pyknotic changes induced by mtx in the 
 present study are caused by apoptosis . in the present study ,
pyknotic neuroepithelial cells 
 in the telencephalon at 6 hr were positive for cleaved caspase-3 , but negative for tunel 
 staining .
this result may reflect that the cleavage of caspase-3 precedes dna fragmentation 
 in the process of the neuroepithelial cell apoptosis induced by mtx .
the previous studies showed that dna - damaging chemical administration at gd 12 or 13 
 induces apoptosis in neuroepithelial cells of the telencephalic wall of fetal brain [ 19,20,21 , 28 , 30 , 39,40,41,42 ] .
these apoptotic neuroepithelial cells are mainly 
 observed in the dorsal layer after treatment with enu [ 6 , 19,20,21 ] in the medial to dorsal layer after 
 treatment with hydroxyurea , 5-fu [ 6 , 42 ] , busulfan 
  and etoposide   and in the ventral to medial layers after treatment with 5azc [ 6 , 39 , 40 ] . in the present study , apoptotic neuroepithelial 
 cells localized throughout all layers of the telencephalic wall , but their distribution was 
 different from those of apoptotic neuroepithelial cells induced by the above - mentioned other 
 dna - damaging chemicals ( table 4table 4.location of apoptotic cells in the ventricular zone of telencephalon after 
 treatment with dna - damaging chemicalslocation of apoptotic cells in the ventricular zone of 
 telencephalonethylnitrosoureadorsal layerhydroxyurea , busulfan , etoposide , 5-fluorouracilmedial to dorsal layers5-azacytidineventral to medial layersmethotrexateall layers ) .
it is known that in the ventricular zone of fetal brain , the positions of 
 the nuclei of neuroepithelial cells are correlated with their cell cycle phase .
s - phase nuclei are located in the dorsal layer of the 
 ventricular zone .
they migrate inward during the 
 g2 phase , and mitosis occurs at the ventricular surface .
then , they migrate outward during the g1 phase and enter the s phase again 
 . in brief , the nuclei during the g1 or g2 phase 
 are in the middle layer of the ventricular zone .
the distribution of apoptotic 
 neuroepithelial cells throughout all layers of the telencephalic wall in the present study 
 may reflect that mtx induces apoptosis of neuroepithelial cells in all phases of the cell 
 cycle or that apoptosis occurs independently of cell cycle arrest . whereas a previous study 
 showed that apoptosis of the human hepatoma hepg2 cells induced by folate deficiency was 
 specific for s- and/or g2-phase arrest
, the 
 present result suggests that mtx induces apoptosis of neuroepithelial cells through a 
 mechanism other than s- and/or g2-phase arrest . in the present study , the pyknotic , tunel and cleaved caspase-3 indices of neuroepithelial 
 cells in the telencephalon peaked 36 hr after treatment with mtx at gd 13 ( table 5table 5 .
timing of the maximal level of cell death indices and minimal level of cell 
 proliferation indices in the telencephalon after dna - damaging chemicals 
 treatmentmaximal level of cell death indicesminimal level of cell proliferation indicespyknosistunelcleaved caspase-3mitosisphospho - histone h31--d - arabinofuranosylcytosine9 hrethylnitrosourea12 hrt-2 toxin12 hr5-azacytide12 hr24 hr 
busulfan48 hr48 hr6-mercaptopurine36 hr36 hrmethotrexate36 hr36 hr36 hr636 hr636 hr ) . the index of pyknotic cells in the telencephalon peaked 912 hr after 
 treatment with ara - c   and enu .
the index of tunel - positive cells peaked 12 hr after 
 treatment with t-2 toxin and 5azc [ 34 , 39 ] , and the index of cleaved caspse-3-positive cells 
 peaked 48 hr after treatment with busulfan .
on 
 the other hand , in the present study , mitotic and phospho - histone h3 indices in 
 telencephalon treated with mtx decreased significantly at 6 hr and maintained that low level 
 throughout the experimental period ( table 5 ) . 
 the mitotic cell index in the telencephalon significantly decreased at 30 hr and reached its 
 lowest level 36 hr after treatment with 6-mp , 
 while the mitotic cell index peaked 6 hr , decreased thereafter and reached the minimal level 
 24 hr after treatment with 5azc .
the index of 
 phospho - histone h3-positive cells significantly decreased at 24 hr and reached its minimal 
 level 48 hr after treatment with busulfan , while 
 it reached the lowest level 36 hr after treatment with 6-mp .
the distribution of pyknotic neuroepithelial cells and the time - course 
 changes of the indices of pyknotic and mitotic neuroepithelial cells in the telencephalic 
 wall were different from those of the previously - mentioned other dna - damaging chemicals . 

these differences may reflect the disparity in mechanisms of apoptosis and the inhibition of 
 cell proliferation in neuroepithelial cells among dna - damaging chemicals .
further 
 investigations should be conducted to clarify the true cause of these differences . in the present study
, mtx induced fewer pyknotic changes of neuroepithelial cells in the 
 metencephalon than in other brain regions .
the result of a previous study suggested that 
 cell - proliferative activity was associated with the sensitivity to mtx .
the different pyknotic indices of the mtx - treated 
 group among the brain regions in the present study may reflect the disparity of 
 cell - proliferative activity . in a previous study ,
mtx inhibited the development of individual brain parts to varying degrees in 
 chick embryos .
the same study suggested that the different effects of mtx on various brain 
 parts may be due to different schedules of cell proliferation among individual brain parts . 

other previous studies demonstrated that busulfan or 5-fu treatment at gd13 in rat fetus 
 induced pyknotic changes of neuroepithelial cells in metencephalon to the same degree as 
 those in diencephalon and mesencephalon [ 29 , 42 ] .
these results suggest that there are differences in 
 sensitivities to mtx , busulfan and 5-fu of neuroepithelial cells in the metencephalon .
there are several studies describing brain malformation induced by mtx in human and rabbits 
 [ 2 , 16 , 35 ] . a report of seidahmed et al .  
described that 2.5 mg of mtx 3 times a day for 7 
 days , for a total of 52.5 mg at 6 weeks of gestation , induced alobar holoprosencephaly , 
 cerebellar hypoplasia and agenesis of the corpus callosum in human .
reported that mtx 5 mg / day 
 treatment for 14 days at the 5th week post - conception induced brain anomalies including 
 semilobar holoprosencephaly and hydrocephalus in human . in rabbits , mtx 19.2 mg / kg 
 administration during gd 1015 induced various anomalies including hydrocephalus . in the present study , single dose - administration of 
 mtx 30 mg / kg on gd 13 induced fetal death 48 hr after treatment , and the cause of fetal 
 death in the present study may be relevant to dose , number and timing of mtx 
 administration . in conclusion ,
mtx administration of 30 mg / kg on gd 13 induced apoptosis of neuroepithial 
 cells and inhibited mitosis of these cells in telencephalon 636 hr after treatment .
the 
 distribution of apoptotic neuroepithelial cells in the telencephalic wall and the whole 
 brain and the time - course changes of the indices of apoptotic and mitotic neuroepithelial 
 cells were different from other dna - damaging chemicals reported previously .
while the 
 detailed mechanisms of mtx - induced neuroepithelial cell damage in fetal brain remain 
 unclear , the present results provide fundamental information about the fetal brain damage 
 induced by mtx .
the present results serve to elucidate the pathogenesis of nervous system 
 anomalies resulting from failed pregnancy termination with mtx or when mothers who are 
 taking mtx for medical reasons become pregnant inadvertently . to our knowledge , this is the 
 first report demonstrating histopathological findings of fetal brain damage induced by 
 mtx .
 abstractpregnant rats were treated with 30 mg / kg of methotrexate ( mtx ) on gestation day 
 13 , and fetal brains were examined histopathologically from 6 to 48 hr after the 
 treatment . in the telencephalon of the control group , there were few pyknotic 
 neuroepithelial cells throughout the experimental period . 
 six hr after mtx treatment , 
 several pyknotic neuroepithelial cells scattered throughout the telencephalic wall . at 
 1236 hr , 
 pyknotic neuroepithelial cells increased significantly and were diffusely 
 distributed throughout the telencephalic wall . 
 neuroepithelial cells were eliminated and 
 showed sparse cell density at 36 hr in the telencephalon . 
 almost all fetuses died at 48 
 hr . 
 most of the pyknotic neuroepithelial cells were positively stained by the tunel method 
 and positive for cleaved caspase-3 . 
 while mitotic and phospho - histone h3-positive 
 neuroepithelial cells were located along the ventricular layer of telencephalon in the 
 control group , they were rarely observed in the same region at 636 hr in the mtx - treated 
 group . 
 mtx induced few pyknotic changes to neuroepithelial cells in the metencephalon , 
 compared to other parts of brain . 
 the distribution of apoptotic neuroepithelial cells and 
 the time - course changes of the indices of apoptotic and mitotic neuroepithelial cells were 
 different from those of other dna - damaging chemicals reported previously . 
 the difference 
 may reflect the disparity in mechanisms of apoptosis and the inhibition of cell 
 proliferation in neuroepithelial cells induced by mtx . to our knowledge , this is the first 
 report demonstrating histopathological findings of fetal brain damage induced by mtx . 
MATERIALS AND METHODS
RESULTS
DISCUSSION