Genetic susceptibility in the neural tube defects induced by ochratoxin A in the genetic arhinencephaly mouse, Pdn/Pdn

It is well known that ochratoxin A (OTA) induces neural tube defects (NTDs) in mice. In the present study, OTA was administered to the genetic polydactyly/arhinencephaly mouse (Pdn/Pdn) to investigate the synergistic effect between gene and environmental toxin. OTA treatment on day 7.5 of gestation increased NTDs in the Pdn/Pdn mouse. The responsible gene for Pdn/Pdn is Gli3. So, it was speculated that specific susceptibility for OTA in the Pdn/Pdn mouse embryo may be due to the severe depression of Gli3 gene expression. As correlated genes, Gli3, Shh and Fgf8 gene expressions were examined in the Pdn mouse embryo on day 9 of gestation after administration of OTA on day 7.5. No alteration of Shh expression was observed in the non-treated Pdn/Pdn, and OTA-treated +/+ and Pdn/Pdn. Fgf8 signal was observed at the anterior neural ridge (ANR) in the non-treated +/+, and that was elongated in the non-treated Pdn/Pdn, and further elongated and more intensive in the OTA-treated Pdn/Pdn. It was suggested that Fgf8 gene expression was affected by the depression of Gli3, and alteration of Fgf8 gene expression was accelerated by the toxicity of OTA in the Pdn/Pdn.     

Gender-dependent differences in the incidence of ochratoxin A-induced neural tube defects in the Pdn/Pdn mouse

Genetic polydactyly/arhinencephaly mouse embryo, Pdn/Pdn , exhibits suppression of Gli3 gene expression. Ochratoxin A (OTA) is a teratogen that causes neural tube defects (NTD) in mice. We investigated gender-dependent differences in the incidence of NTD induced by OTA in the Pdn/Pdn mouse. After administering 2 mg/kg OTA to Pdn /+ female mice, mated with Pdn /+ males, on day 7.5 of gestation, we examined the genotypes, sex and NTD of fetuses on day 18. Non-treated Pdn / Pdn had a 15.8% risk of NTD, and all NTD fetuses were female. When Pdn / Pdn embryos were exposed to OTA, the incidence of NTD increased to 16 (51.6%) of 31 Pdn / Pdn fetuses, and 10 (71.4%) of 14 male Pdn / Pdn fetuses exhibited NTD. From these results, it was speculated that NTD in OTA-treated male Pdn / Pdn were due to the synergistic effect between depressed Gli3 and altered sex-correlated gene expression from OTA treatment. After treatment with OTA, the embryos were recovered on day 9 and gene expressions, which were correlated with Gli3 , telencephalic morphogenesis, formation of gonadal anlage, and gender-dependent differentiation were investigated. From real-time polymerase chain reaction analysis results, it was suggested that the manifestation of NTD in the male OTA-treated Pdn/Pdn might be due to the complicated altered gene expressions among Gli3, Wnt7b, Wnt8b , Fez1 , Barx1, Lim1, Dmrt1, Igf1 , Fog2, Dax1 and Sox9, and in particular, upregulation and gender-dependent difference in Barx1 and gender-dependent difference in Sox9 gene expressions might be noteworthy findings.     

Exencephaly induction by valproic acid in the genetic polydactyly/arhinencephaly mouse, Pdn/Pdn

Non-treated homozygous polydactyly/arhinencephaly (Pdn/Pdn) mouse fetuses exhibited exencephaly in 16.7% of cases. Treatment of Pdn/Pdn mice with 350 mg/kg of valproic acid (VPA) on days 8.5 and 9.5 of gestation increased the rate of exencephaly to 66.7%. The responsible gene for the Pdn mouse phenotype has been determined to be Gli3, and the suppression of Gli3 gene expression has been documented in Pdn/Pdn embryos. We investigated how the sonic hedgehog (Shh) and Fgf8 genes, the correlated genes of Gli3, are expressed in the VPA-treated exencephalic Pdn/Pdn embryos on day 10 of gestation, using whole mount in situ hybridization (WISH) and real-time PCR methods. We could not detect any alterations in Shh expression by real-time PCR, or WISH in the non-treated Pdn/Pdn and VPA-treated exencephalic Pdn/Pdn embryos. Altered Fgf8 expression patterns were observed in the commissural plate and dorsal isthmal neuroepithelium in the non-treated Pdn/Pdn embryos. We speculated that the altered expression of Fgf8 might be the result of down-regulation of Gli3 in Pdn/Pdn embryos. Fgf8 gene expression in the commissural plate and dorsal isthmal neuroepithelium exhibits wide or altered signal patterns in the VPA-treated exencephalic Pdn/Pdn embryo. From these findings, it was suggested that down-regulation of Gli3 gene expression induced the altered expression of Fgf8 in the Pdn/Pdn embryos, and that VPA treatment accelerated the alterations of Fgf8 gene expression in the Pdn/Pdn embryos. It was further speculated that altered expression of Fgf8 in the commissural plate may be the fundamental cause of exencephaly, and that the synergistic effect between gene and drug shown in this experiment may explain the differences of sensitivity in the side-effects of the drug.     

Sonic hedgehog expression in Gli3 depressed mouse embryo, Pdn/Pdn

The phenotype of the genetic polydactyly/arhinencephaly mouse (Pdn/Pdn) is similar to Greig cephalopolysyndactyly syndrome (GCPS), which is induced by mutation of GLI3. Suppression of Gli3 gene expression has been observed in Pdn/Pdn. Thus, the gene responsible for Pdn/Pdn has been considered to be Gli3. Recently, the mutation point was demarcated, that is, a transposon was inserted into intron 3 of the Gli3 gene in the Pdn mouse. Forward and reverse primers were constructed in intron 3 near the insertion point. A forward primer in the long terminal repeat region of the transposon was also constructed. Now we can discriminate +/+, Pdn/+, Pdn/Pdn embryos from the PCR products. After genotyping of the Pdn embryos, Gli3 and other correlated gene expressions, such as sonic hedgehog (Shh), Bmp-2, Bmp-4, ptc-1, were analyzed by real-time PCR method. Gli3 gene expression in Pdn/Pdn was suppressed to 20-30% of +/+, and that in Pdn/+ was about 60% of +/+ through all the embryonic and neonatal periods examined. As Shh has been considered to be an antagonist of Gli3, Shh expression was analyzed, and a difference among genotypes was observed only on day 9 of gestation. We could not detect any alterations among genotypes in other gene expressions examined. Gli3 and Shh gene expression were also analyzed on day 9 by whole-mount in situ hybridization in the +/+ and Pdn/Pdn embryos. Neuroectoderm was positive by Gli3 probe in +/+ but not in Pdn/Pdn. Notochord, floor plate and prechordal mesoderm were positive by Shh probe both in +/+ and Pdn/Pdn embryos, but ectopic and/or over-expression of Shh were not observed in Pdn/Pdn embryos.     

Integration of a transposon into the Gli3 gene in the Pdn mouse

ABSTRACT  The phenotype of the genetic polydactyly/arhinencephaly mouse (Pdn/Pdn) is similar to Greig cephalopolysyndactyly syndrome (GCPS), whose responsible gene is GLI3. Suppression of Gli3 gene expression has been observed in the Pdn/Pdn and integration of retrotransposon in Gli3 gene in the Pdn mouse has been reported. Thus, the responsible gene for Pdn/Pdn is thought to be Gli3 , but the site of mutation within the gene has not been demarcated.
In the present study, we demonstrated that 5442 bp of early retrotransposon was inserted into intron 3 of Gli3 gene in the Pdn mouse (Gli3^Pdn). This transposon had almost the same sequence as MMY17106 (EMBL). It had 317-bp long terminal repeat at both ends followed by the identical 6-bp target duplication sequence, GAGACT. Forward and reverse PCR primers were constructed in intron 3 near the insertion point, and a forward primer in the transposon was also constructed. These primers allowed us to discriminate +/+, Pdn /+ and Pdn/Pdn embryos by the PCR products. Morphological determination of the genotypes in the Pdn mouse embryos is impossible before day 12 of gestation. Quick discrimination method of genotypes developed in the present study allows us to investigate the early dysmorphogenetic mechanisms in the brain and limbs in the Pdn/Pdn embryos. Then, the dysmorphogenetic mechanisms in the Pdn/Pdn may be extrapolated to those in GCPS.     

The Role of Apoptosis in the Manifestation of Polydactyly and Arhinencephaly in Genetic Mutant Mouse Pdn/Pdn*

Abstract Pdn/Pdn mouse exhibits preaxial polydactyly and arhinencephaly, including various brain abnormalities such as the absence of corpus callosum and hydrocephaly. We have investigated the mechanism of the manifestation of polydactyly. The abolishment of the deep preaxial mesodermal programmed cell death, foyer primaire préxial (fpp), has been considered to be the starting point of the manifestation of preaxial polydactyly. With this hypothesis, we electrocauterized the fpp region of Pdn/Pdn embryos, because Pdn/Pdn lacks fpp. After surgery, Pdn/Pdn embryos were developed using whole embryo culture system or exo utero method. They exhibited 5 digits in their limbs which received surgery in fpp region. Meanwhile, much more apoptotic degenerations were observed in the brains of Pdn/Pdn newborns and embryos than normals. Anti-single-stranded DNA antibody was used to detect the localization of apoptotic cell death in the brains of Pdn/Pdn mice. TRPM-2 gene has been considered to be an indicator of apoptosis. The brains of Pdn/Pdn newborns and embryos showed higher TRPM-2 gene expression in Northern blot analysis. From these results, we speculated that abnormal apoptosis in the periventricular zone induced the expansion of the ventricle followed by hydrocephaly.     

Hydrocephalus manifestation in the genetic polydactyly/ arhinencephaly mouse (Pdn/Pdn)

ABSTRACT  The genetic polydactyly/ariiinencephaly mouse, Pdn/Pdn , exhibits severe polydactyly both in the fore-and hindlimbs, hydrocephalus, and agenesis of the olfactory bulbs, corpus callosum, and anterior commissure. The mechanism of hydrocephalus manifestation in Pdn/Pdn was investigated in the present study. Ink was injected into the left lateral ventricle in the Pdn/Pdn and +/+ newborn mice. After incubation at 32°C for different time intervals, the heads were fixed in Bouin's solution and were subsequently decalcified in 0.5 mol/L of EDTA solution, paraffin sectioned, and stained with hematoxylin and eosin.
Ink spread into the 3rd and right lateral ventricles and flowed to the 4th ventricle and Magendie's foramen rapidly in Pdn/Pdn mice. This rapid spread was due to the dilatation of the interventricular foramen and that the lateral ventricle was directly connected with enlarged 3rd ventricle in Pdn/Pdn. In spite of the rapid spread of ink in the cerebrospinal fluid pathway, ink was not observed in the subarachnoid space around the superior sagittal sinus at 3.5 or 10 hours in Pdn/Pdn mice.
The superior sagittal sinus was narrower in Pdn/Pdn than in +/+, and the arachnoid villi were not observed in Pdn/Pdn. From these observations, we suggested that absorption of cerebrospinal fluid from the arachnoid villi in the superior sagittal sinus stagnated and that stagnation of the fluid in the ventricles was the cause of hydrocephalus in Pdn/Pdn mice.     

Prevention of Genetic Polydactyly in Polydactyly Nagoya (Pdn) Mice in Vitro by Surgical Treatment of Foot Plate during Embryogenesis

Abstract Pdn/Pdn fetuses show preaxial Polydactyly of duplicated or triplicated metacarpal/metatarsal type in the fore- and hindlimbs. Pdn /+ fetuses show one extra digit of distal phalangeal type preaxially in the hindlimb and deformity of distal phalanx of the 1st digit in the forelimb. Normal patterns of physiological cell death in the preaxial apical ectodermal ridge (AER) and the deep preaxial mesoderm (fpp) were disrupted in Pdn/Pdn embryos. It was supposed that delayed involution of preaxial AER might have caused the abolishment of fpp, which in turn could have induced polydactyly in Pdn/Pdn .
In order to induce cell death in the fpp region artificially, tissue destruction of the fpp region of right fore- and hindfoot plates of Pdn/Pdn , Pdn /+ and +/+ embryos was performed by an electric knife on day 11.5 of gestation, and the embryos were cultured in the rotator culture system for 20 hours. The non-treated left foot plates served as the controls.
In Pdn/Pdn embryos, the non-treated left foot plates showed abnormal protuberance and/or the extra digital rays preaxially. But the treated right foot plates did not exhibit these abnormal characteristics. Instead they revealed 5 digital rays of mesodermal condensation in the histological sections.
These results indicated that the restriction of the artificial tissue destruction in the fpp region could prevent the manifestation of preaxial extra digits in Pdn mice.     

Phenotypic differences in the brains and limbs of mutant mice caused by differences of Gli3 gene expression levels

ABSTRACT  The genetic polydactyly/arhinencephaly mouse, Pdn/Pdn , exhibits severe polydactyly both in the fore-and hindlimbs, agenesis of the olfactory bulbs, corpus callosum, anterior commissure, and hydrocephalus. A candidate gene for the Pdn mouse has been speculated to be Gli3 , because Pdn has been considered to be an allele of Xt whose responsible gene has been clarified to be Gli3. Recently, it has been cleared that retro-transposons are inserted into nitron 3, upstream of zinc finger domain, of the Gli3 gene in the Pdn mouse, resulting to the severe suppression of Gli3 gene expression in Pdn/Pdn embryos. Meanwhile, Xt^J/Xt^J mice exhibit more severe polydactyly than that of Pdn/Pdn. Arhinencephaly and microholoprosencephaly including agenesis of the olfactory bulbs, corpus callosum, anterior commissure, hippocampal commissure, habenular commissure, and posterior commissure, and moreover, the cerebral cortical plates and hippocampus are not formed in the Xt^J/Xt^J mice. The Xt^J/Xt^J mouse has a large deletion in Gli3 structural gene and shows null expression. From these corroborations, we speculated that the differences in the Gli3 gene expression levels resulted in the phenotypic differences between the Pdn/Pdn and Xt^J/Xt^J mice.     

Suckling dysfunction caused by defects in the olfactory system in genetic arhinencephaly mice

Mouse newborns find their mother's nipples and suckle milk by themselves. It has been argued which sense organ they use when locating their mother's nipples to suckle milk. Olfactory or tactile sensory systems are primary candidates. In the present study, we investigated the trigeminal-whisker sensory and olfactory systems in genetic arhinencephaly mouse embryos (Pdn/Pdn). Pdn/Pdn newborns do not suckle milk and die within 1 day after birth. Dysfunction of nipple-searching behavior was clear in Pdn/Pdn newborns. Pdn/Pdn newborns had a complete developmental failure in the olfactory nerve projection to the central nervous system and no olfactory bulb architecture. The trigeminal-whisker system was intact in this strain. From the results of these experiments, it was suggested that the olfactory system is essential for nipple-searching behavior and suckling milk and that the trigeminal-whisker system is not able to substitute for the lack of olfactory input in mouse newborns.     

Amelioration of sodium valproate-induced neural tube defects in mouse fetuses by maternal folic acid supplementation during gestation

Infants of epileptic women treated with valproic acid (VPA) during pregnancy have a higher risk of developing spina bifida than those of the general population. VPA induces exencephaly in experimental animal embryos. But the pathogenetic mechanism remains rather elusive. Antiepileptic drugs (AED) in general accentuate pregnancy-imposed fall in maternal folate levels. Periconceptional folic acid supplementation is reported to protect embryos from developing neural tube defects (NTD). Conflicting results have been reported by experimental studies that attempted to alleviate VPA-induced NTD by folic acid. Our objectives were to determine the critical developmental stages and an effective dose of folic acid for the prevention of VPA-induced exencephaly in mouse fetuses. A single teratogenic dose of 400 mg/kg of VPA was administered to TO mice on gestation day (GD) 7 or 8. It was followed by (1) a single dose of 12 mg/kg of FA (folinic acid) or (2) 3 doses of FA 4 mg/kg each. In experiment (3), FA (4 mg/kg) was administered thrice daily starting on GD 5 and continued through GD 10. These animals received VPA on GD 7 or 8. VPA and B12 concentrations were determined by radioimmunoassay. The single heavy dose of FA had no rescue effect on NTD. Three divided doses of FA on GD 7 and continuous dosing of FA from GD 5 through GD 10 substantially reduced the VPA-induced exencephaly in the fetuses. In the later experiments, the neural folds elevated faster than the non-supplemented group. VPA considerably reduced maternal plasma folate and B12 concentrations. The heavy dose of FA only moderately improved vitamin levels. Three divided doses of FA elevated the vitamin levels slightly better but it was the prolonged dosing of FA that was associated with sustained elevation of plasma levels higher than the control levels and acceleration of neural tube closure thus accounting for the pronounced protection against VPA-induced NTD development. These data suggest that plasma levels of FA and B12 have to be kept substantially elevated and maintained high throughout organogenesis period to protect embryos against VPA-induced NTD in this mouse model.     

Birth defects caused by mutations in human GLI3 and mouse Gli3 genes

GLI3 is the gene responsible for Greig cephalopolysyndactyly syndrome (GCPS), Pallister–Hall syndrome (PHS) and Postaxial polydactyly type-A (PAP-A). Genetic polydactyly mice such as Pdn/Pdn (Polydactyly Nagoya), Xt^H/Xt^H (Extra toes) and Xt^J/Xt^J (Extra toes Jackson) are the mouse homolog of GCPS, and Gli3^tmlUrtt/Gli3^tmlUrt is produced as the mouse homolog of PHS. In the present review, relationships between mutation points of GLI3 and Gli3 , and resulting phenotypes in humans and mice are described. It has been confirmed that mutation in the upstream or within the zinc finger domain of the GLI3 gene induces GCPS; that in the post-zinc finger region including the protease cleavage site induces PHS; and that in the downstream of the GLI3 gene induces PAP-A. A mimicking phenomenon was observed in the mouse homolog. Therefore, human GLI3 and mouse Gli3 genes have a common structure, and it is suggested here that mutations in the same functional regions produce similar phenotypes in human and mice. The most important issue might be that GCPS and PHS exhibit an autosomal dominant trait, but mouse homologs, such as Pdn/Pdn , Xt^H/Xt^H , Xt^J/Xt^J and Gli3^tmlUrt/Gli3^tmlUrt , are autosomal recessive traits in the manifestation of similar phenotypes to human diseases. It is discussed here how the reduced amounts of the GLI3 protein, or truncated mutant GLI3 protein, disrupt development of the limbs, head and face.     

Effects of thalidomide on Fgf8, Bmp4 and Hoxa11 expression in the limb bud in Kbl:JW rabbit embryos

Thalidomide (TM) induces limb defects in humans and some animal species including rabbits. Although the mechanism of TM‐induced limb defects has been investigated for a long period, the limb development‐related genes expressions have not been vigorously characterized in rabbits. In this study, we investigated the Fgf8, Bmp4 and Hoxa11 expressions in TM‐treated JW rabbit embryos on gestation days (GDs) 10, 11 and 12 by whole mount in situ hybridization. On GDs 10 and 11, growth retardation of the embryo was induced by TM treatment. The Fgf8 expression lengths on GDs 10 and 11 in the forelimb bud were significantly or tended to be decreased in the TM‐treated embryos, which was correlated to the growth retardation and was not considered to be directly relevant to the teratogenic effect of TM in the forelimb. The TM‐induced characteristic changes in the expression pattern of Hoxa11 and Bmp4 on GDs 10 and/or 11 were not noted. On GD 12, TM‐induced growth retardation was not noted and the Fgf8 and Bmp4 expressions were not changed. On the contrary, Hoxa11 expression was narrowed at the anterior region, which was located on the radial side, and was not changed at the middle and posterior regions in the forelimb bud and in all regions in the hindlimb bud. Because the radius malformations were induced by TM treatment, we concluded the decrease in the Hoxa11 expression was related to the TM‐induced limb defects and can be a good marker for early prediction of the teratogenic effect of TM.     

肛门直肠畸形胎鼠直肠末端Gli2、BMP4基因的表达

目的探讨乙烯硫脲（ETU）致畸胎鼠直肠末端Gli2、BMP4基因在胎鼠直肠肛门及其畸形发生过程中的表达和作用。方法取妊娠SD大鼠30只,按受孕时间配对分成2组,实验组（n=15）于孕10d灌胃注入10g／LETU （125mg／kg）,对照组n=15）予等量蒸馏水。两组分别于孕13d、14d、15d、16d和17d剖宫取胎鼠直肠末端1cm提取RNA,采用RT—PCR和RealtimePCR法检测Gli2、BMP4基因在直肠肛门发育不同时期的表达情况。结果采用RT-PCR法检测Gli2、BMP4基冈在直肠肛门发育不同时期的表达情况．发现在胎鼠第13～17天,实验组直肠末端Gli2、BMP4mRNA的表达水平均明显低于正常胎鼠直肠末端（P〈0．05）。而Realtime PCR检测Gli2、BMP4mRNA表达时,发现实验组Gli2的表达在胚胎第13天、第14天、第15天分别为（0．73＋0．07、0．60＋0．09、0．81＋0．06）,勺对照组相比显著降低（P〈0．05）,而在孕第16天、第17天实验组Gli2表达比对照组虽有下降,但无统计学意义;实验组BMP4的表达在孕第3～17天均较对照组下降,差异有统计学意义。结论直肠肛门的正常发育需要Shh信号通路的正常表达,Shh信号通路中转录因子Gli2和靶基囚BMP4的异常表达在肛门直肠畸形发生过程中起重要作用,ETU可能影响Shh信号转导通路和肛门直肠畸形发生。     

Morphological analysis of neural tube defects: Chlorambucil-induced exencephaly in mice

Abstract: Exencephaly has been induced in mouse embryos by chlorambucil (CA), a cytotoxic agent. To understand the course of development of this malformation, open neural tube defects in CA-treated mice were examined using light and scanning electron microscopes (SEM). CA was given to the pregnant mice on day 7.4 of gestation. Embryos were removed and fixed on gestational days 9.3–9.4, 9.7–9.8 and 10.4, and compared to control embryos from untreated mice. By gestational day 9.4, all control embryos had closed neural tubes, except for the posterior neuropores, and well developed brain vesicles. By contrast, in the experimental embryos the frequencies of open neural tube were 26/33 (78.8%), 32/ 40 (80.0%) and 23/66 (34.8%) on each day examined, respectively. Open neural tube defects were classified into six patterns according to the location and magnitude of the open area. The patterns of open neural tube on day 9.7–9.8 were diverse; however, in almost all cases on day 10.4 the open neural tube appeared in a region from the caudal forebrain to the rostral hindbrain. It was evident that an unusual pattern of closure of the neural tube was involved in forming the cranial neural tube. The present study shows that failure of closure of the cranial neural tube in the CA-treated mouse embryos can be defined as a primary neural tube defect (NTD), which can be, in part, repaired by unusual closure of the neural tube.
At high magnifications of SEM, the neuroectodermal surfaces of the 9.0-day affected embryos often had a number of slender processes projecting from the neuroepithelial cells. "Ruffles" and "blebs" at the lateral edges of the neural folds were observed in both control and affected embryos.     

Arsenate-induced neural tube defects not influenced by constant rate administration of folic acid

Serious suggestions have been made that dietary supplementation with folic acid (FA) and perhaps other vitamins during pregnancy may reduce the incidence of neural tube defect (NTD) in human newborns. The purpose of these experiments was to evaluate the effect of continuous infusion of FA on the incidence of NTDs induced by arsenate. This teratogen induces NTDs in up to 90% of golden hamster fetuses when administered acutely during critical stages of embryogenesis. FA was administered by subcutaneously implanted osmotic minipumps beginning on the 6th day of gestation, 48 h before an acutely administered dose of sodium arsenate. The protective effect of FA was examined at three teratogenic dose levels of arsenate: optimal, with 905 NTDs, intermediate, with 38% NTDs, and low, with 20% NTDs. Fetuses were recovered at day 13 of gestation and examined for NTDs and other malformations. Maternal red cell folate levels were determined on day 8, 48 h after implantation of the pumps. The results show that the maternal red blood cell level of FA can be significantly increased within 48 h by chronic infusion to levels which are almost two times (550 ng/ml) control levels. There was no significant protection against arsenate-induced NTDs following FA supplementation at any of three levels of this teratogen.     

鸡胚神经管缺陷脊髓病理与组织化学研究

目的 鸡胚内注射甲氨喋呤（ＭＴＸ）,制作鸡神经管缺陷动物模型。利用该模型,进一步研究观察病变的病理变化及组织化学改变。方法 选用上海农科院“莱杭”鸡受蛋６６校,在孵化期第４、５天,注射ＭＴＸ,计量为：０．０１ｍｇ／ｋｇ及０．０２ｍｇ／ｋｇ。孵化３周后,选取病变鸡,采用ＮＡＤＰＨ－黄麦组织化学染色,测定变脊髓神经元ＮＯＳ含量;电镜观察病变脊髓神经元内线粒体结构变化。结果 ＮＡＤＰＨ－黄递酶组织化学染