Xue-Zhi Sun

An Immunohistochemical Study of Radial Glial Cells in the Mouse Brain Prenatally Exposed to γ-irradiation

The features of a glial cell population in the developing brain of mice prenatally exposed to 60Co γ-irradiation at the most radiosensitive stage were studied with immunohistochemistry for anti-midkine (MK), anti-vimentin (Vim), and anti-GFAP antibodies. Anti-MK– and anti-Vim–positive radial glial fibers distributed in a similar radial fashion; these fibers were observed primarily in the embryonic period and disappeared after birth. Anti-MK– and anti-Vim–stained radial fibers ran perpendicular to the pial surface in controls, whereas such fibers were disorganized 6 hours (h) after irradiation. This finding provided new evidence that the migratory pathways of young neurons were interrupted beginning a few hours after irradiation. By E17 the ectopic cell masses formed so as to replace the parts of the ventricular zone where no anti-MK immunoreactive radial fibers were present, but where anti-GFAP–stained fibrillary astrocytes emerged in the ectopic cell masses from the early postnatal period. The results suggested a twofold source of the generated astrocytes: either directly from a separate precursor of the astrocytes, or due to the transformation of the classic radial glial cells. In the newborn, numerous protoplasmic transitional forms displaced by astrocytes in irradiated brains indicated that reactive gliosis was a powerful response of a brain exposed to irradiation.

Abnormal expression of tyrosine hydroxylase immunoreactivity in cerebellar cortex of ataxic mutant mice

Expression of tyrosine hydroxylase (TH) was examined immunohistochemically in the cerebellum of two ataxic mutants, Rolling mouse Nagoya (RMN) and dilute-lethal mice (DL). In littermate controls of both mutants, a few TH-positive Purkinje cells were distributed sparsely and their number was smaller than in the mutants at any ages examined. In RMN, TH-positive Purkinje cells were distributed in lobule IX and X, and were arranged into parasagittal bands at 2 weeks of age. TH-positive Purkinje cells increased in number and were widely distributed throughout the vermis at 3 weeks of age. In adult RMN, TH-positive Purkinje cells were found in all lobules of the cerebellum. Their parasagittal bands also became evident in the hemisphere. In DL, TH-positive Purkinje cells were mainly distributed in vermal lobules IX and X, and the flocculus at 3 weeks of age. They were also found as bands in lobules IX and X. The results suggest that abnormal expression of TH in Purkinje cells may not be specific to the allelic group. Since TH promoter is activated by Ca2+, TH expression in the mutant Purkinje cells may predict neuronal dysfunction caused by alterations in cellular Ca2+ currents.

Purkinje cell degeneration in mice lacking the xeroderma pigmentosum group G gene

Laboratory mice carrying the nonfunctional xeroderma pigmentosum group G gene (the mouse counterpart of the human XPG gene) alleles have been generated by using gene‐targeting and embryonic stem cell technology. Homozygote animals of this autosomal recessive disease exhibited signs and symptoms, such as postnatal growth retardation, reduced levels of activity, progressive ataxia and premature death, similar to the clinical manifestations of Cockayne syndrome (CS). Histological analysis of the cerebellum revealed multiple pyknotic cells in the Purkinje cell layer of the xpg homozygotes, which had atrophic cell bodies and shrunken nuclei. Further examination by an immunohistochemistry for calbindin‐D 28k (CaBP) showed that a large number of immunoreactive Purkinje cells were atrophic and their dendritic trees were smaller and shorter than in wild‐type littermates. These results indicated a marked degeneration of Purkinje cells in the xpg mutant cerebellum. Study by in situ detection of DNA fragmentation in the cerebellar cortex demonstrated that some deoxynucleotidyl transferase (TdT)‐mediated dUTP‐biotin in situ nick labeling (TUNEL)‐positive cells appeared in the granule layer of the mutant mice, but few cell deaths were confirmed in the Purkinje layer. These results suggested Purkinje cell degeneration in the mutant cerebellum was underway, in which much Purkinje cell death had not appeared, and the appearance of some abnormal cerebellar symptoms in the xpg‐deficient mice was not only due to a marked Purkinje cell degeneration, but also to damage of other cells. J. Neurosci. Res. 64:348–354, 2001.

DIFFERENT PATTERNS OF ABNORMAL NEURONAL MIGRATION IN THE CEREBRAL CORTEX OF MICE PRENATALLY EXPOSED TO IRRADIATION

A characteristic abnormal cortical architecture in the adult brain was produced in mice subjected to 1.5 Gy of X-irradiation on embryonic day 14. Neurons in the lateral regions were organized into an essentially six-layered structure, while neurons in the dorsal regions formed a unique four-layered cortex. The patterns of neuronal migration in these different cortical regions were examined with immunohistochemistry for anti-bromodeoxyuridine (BrdU), anti-midkine (MK), and anti-glial fibrillary acidic protein (GFAP) antibodies. In the cortical lateral region, BrdU-labeled cells in the upper layers were fewer, and those in lower layers more numerous in prenatally irradiated mice than in control, while in the dorsal region (four-layered region), BrdU-labeled cells were very few in layer 2, and a large number of labeled-cells remained in layer 4. These results indicated that some neuroblasts in the lateral cortical region could not migrate to the upper layers, and that most neuroblasts in the dorsal cortical region failed to pass through the earlier migration zone. MK- and GFAP-stained radial glial fibers showed that the radial fibers were consistently oriented in the direction of neuronal migration in the control brains. However, in the irradiated brain, such radial fibers were crumpled in the lateral region, or were reduced markedly in number in some parts of the dorsal region. These results revealed that neuronal migratory pathways (radial glial fibers) were destroyed differently in different regions, and that X-rays killed some cells including radial glial cells or their precursors during the embryonic stage. These effects of radiation on the developing brain may result from the possibility that neurogenetic time is different or there are cellular mechanisms involved in the radiosensitivity among different regions.

Radiation-induced tissue abnormalities in fetal brain are related to apoptosis immediately after irradiation

PURPOSE To investigate the relation between the incidence of radiation-induced tissue abnormalities in fetal brain and the extent of p53-dependent apoptosis. MATERIALS AND METHODS Pregnant mice with wild-type p53(+/+), heterozygous p53(+/-) and homozygous mutant p53(-/-) fetuses received whole-body X-irradiation on day 13 of gestation. The extent of apoptosis 6 hr after irradiation and the incidence of tissue abnormalities 3 days after irradiation in the brain were evaluated by histological examination of brain mantle. RESULTS The percentage of apoptotic cells increased linearly with dose in p53(+/+) and p53(+/-) fetuses, but no increase was found in p53(-/-). Approximately twice the dose was necessary in p53(+/-) fetuses to induce an apoptotic response to the extent observed in p53(+/+). Fetuses with brain-tissue abnormalities, such as a destroyed ventricular lining and rosettes with a central hollow appeared at a dose of 1.5 and 3.0 Gy, and the incidence was markedly increased following a dose of 2.25 and 3.75Gy in p53(+/+) and p53(+/-) mice, respectively, but no fetus with tissue abnormalities appeared in p53(-/-) at up to 3.75 Gy. Approximately twice the dose was necessary in p53(+/-) fetuses to induce brain-tissue abnormalities to the extent seen in p53(+/+) mice. CONCLUSION The extent of apoptosis 6 hr after irradiation and the incidence and severity of brain-tissue abnormalities 3 days after irradiation corresponded well, suggesting that radiation-induced tissue abnormalities, such as destroyed ventricular lining, deranged glial fibre and appearance of rosettes in fetal brain were closely related to apoptosis seen 6 hr after irradiation.Purpose : To investigate the relation between the incidence of radiation-induced tissue abnormalities in fetal brain and the extent of p53-dependent apoptosis. Materials and Methods : Pregnant mice with wild-type p53(+/+), heterozygous p53(+/-) and homozygous mutant p53(-/-) fetuses received whole-body X-irradiation on day 13 of gestation. The extent of apoptosis 6 hr after irradiation and the incidence of tissue abnormalities 3 days after irradiation in the brain were evaluated by histological examination of brain mantle. Results : The percentage of apoptotic cells increased linearly with dose in p53(+/+) and p53(+/-) fetuses, but no increase was found in p53(-/-). Approximately twice the dose was necessary in p53(+/-) fetuses to induce an apoptotic response to the extent observed in p53(+/+). Fetuses with brain-tissue abnormalities, such as a destroyed ventricular lining and rosettes with a central hollow appeared at a dose of 1.5 and 3.0Gy, and the incidence was markedly increased following a dose of 2.25 and 3.75 Gy in p53(+/+) and p53(+/-) mice, respectively, but no fetus with tissue abnormalities appeared in p53(-/-) at up to 3.75 Gy. Approximately twice the dose was necessary in p53(+/-) fetuses to induce brain-tissue abnormalities to the extent seen in p53(+/+) mice. Conclusion : The extent of apoptosis 6 hr after irradiation and the incidence and severity of brain-tissue abnormalities 3 days after irradiation corresponded well, suggesting that radiation-induced tissue abnormalities, such as destroyed ventricular lining, deranged glial fibre and appearance of rosettes in fetal brain were closely related to apoptosis seen 6 hr after irradiation.

Abnormal distribution of hippocampal mossy fibers in rats exposed to X-irradiation in utero

The effects of prenatal X-irradiation (0.3, 0.6, 1.2 or 1.8 Gy) on the hippocampal development were examined at six weeks of age in rats. The laminar structure of the hippocampus was deranged in the rats exposed to 0.6 Gy or more. Pyramidal cells in the CA-3 region were more susceptible than those in the CA-1 region. Aberrant mossy fiber terminals were observed in the stratum oriens of the CA-3 region (infrapyramidal mossy fibers) in rats exposed to 0.3 Gy or more.

Ontogenetic pattern of gyrification in fetuses of cynomolgus monkeys

The ontogenetic pattern of gyrification and its relationship with cerebral cortical volume were examined in cynomolgus monkey fetuses. T(1)-weighted coronal magnetic resonance (MR) images at 7 T were acquired from the fixed cerebra of three male fetuses, each at embryonic days (EDs) 70 to 150, and the gyrification index (GI) of each slice was estimated. The mean GI was low (1.1-1.2) during EDs 70 to 90, and then increased dramatically on ED 100. The developmental profiles of the rostrocaudal GI distribution revealed that cortical convolution was more frequent in the parietooccipital region than in other regions during EDs 100 to 150, forming an adult-like pattern by ED 150. The mean GI was closely correlated with the volume of cortical gray matter (r=0.9877), and also with the volume of white matter/intermediate zone (r=0.8961). These findings suggest that cortical convolution is correlated with either the maturation of cortical gray matter or the development of white matter bundles. The characteristic GI distribution pattern of catarrhines was formed by ED 150 in correlation with the progressive sulcal infolding in the parietooccipital region of the cerebrum.

Neurogenesis of heterotopic gray matter in the brain of the microcephalic mouse

Neurogenesis of heterotopic gray matter in the brain of the microcephalic mouse prenatally exposed to X‐rays at embryonic day 13 (E13) was studied immunohistochemically. Bromodeoxyuridine (BrdU) as a marker to label the migrating position of neuroblasts generated at various embryonic stages showed that no “inside‐out” pattern of neuronal migration occurred in the heterotopic cell mass similar to that seen in the laminated cortex. Further results in which midkind (MK) immunoreactive radial glial fibers did not appear in the heterotopic cell mass demonstrated that heterotopia formed in the absence of radial glia system. Different types of cells (pyramidal and non‐pyramidal neurons) in the heterotopic cell mass were identified with immunoreactivity for anti‐parvalbumin and anti‐calbindin D‐28K antibodies in addition to current histological methods. Two major types of neurons were mixed together with random distribution in the heterotopic cell mass. This finding indicates that irradiation might have no selective effects on the precursors of pyramidal and non‐pyramidal neurons. Moreover, anti‐glial fibrillary acidic protein (GFAP) immunostaining showed that numerous astrocytes were present in the heterotopic cell mass. The fact that astrocytes appeared in the heterotopia without the transition from classic radial glial cells to astrocytes suggests that astrocytes might be generated directly from a separate astroglial precursor. J Neurosci. Res. 66:1083–1093, 2001.

Abnormal expression of tyrosine hydroxylase not accompanied by phosphorylation at serine 40 in cerebellar Purkinje cells of ataxic mutant mice, rolling mouse Nagoya and dilute‐lethal

ABSTRACT  This study examined immunohistochemically the expression of an enzymatically active form of tyrosine hydroxylase (TH), phosphorylated TH at Ser40 (phospho‐TH), in the cerebellum of ataxic mutant mice, rolling mouse Nagoya (RMN) and dilute‐lethal (DL). TH immunostaining appeared in some Purkinje cells in RMN and DL, but in a few of the Purkinje cells of littermate controls for both mutants. In all groups of mice, there were no phospho‐TH immunoreactive Purkinje cells in the cerebellum, although the subsets of TH immunoreactive Purkinje cells were found in the adjacent sections. The results suggest that TH expression in the Purkinje cells of ataxic mutants abnormally increases without activation of this enzyme by phosphorylation. This may mean that TH in Purkinje cells is not related to catecholamine synthesis.

Determination of the spatial distribution of major elements in the rat brain with X-ray fluorescence analysis.

An energy dispersive X-ray fluorescence analysis was applied for determining the spatial (two-dimensional) distribution of elemental concentrations in rat brain sections. Freeze-dried brain sections prepared from normal and ischemic rats with middle cerebral artery occlusion were scanned with a collimated X-ray beam (0.18 mm in diameter, 50-kV acceleration voltage). The fluorescent Kalpha X-rays of P, S, Cl, and K were detectable, so that the two-dimensional distribution of fluorescent X-ray intensities could be determined for these elements. Furthermore, quantitative determination was possible for P and K by using the fundamental parameter technique. However, the accurate determination of Na and Ca was difficult, because of the low energy of Kalpha X-ray of Na, and the interference of K-Kbeta with Ca-Kalpha. The change in elemental concentrations in ischemic tissue, including the decrease in K concentration and increase in Cl concentration, was demonstrated by this method as a two-dimensional contour map. Since it is possible to obtain a pictorial representation of the elemental concentration in tissue sections, this method may be useful to evaluate the ionic changes in injured brain tissue in relation to histological or autoradiographical observations.