Kunio Takishima

Neonatal Exposure to Sevoflurane Induces Abnormal Social Behaviors and Deficits in Fear Conditioning in Mice

Background:Neonatal exposure to anesthetics that block N-methyl-d-aspartate receptors and/or hyperactivate &ggr;-aminobutyric acid type A receptor has been shown to cause neuronal degeneration in the developing brain, leading to functional deficits later in adulthood. The authors investigated whether exposure of neonatal mice to inhaled sevoflurane causes deficits in social behavior as well as learning disabilities. Methods:Six-day-old C57BL/6 mice were exposed to 3% sevoflurane for 6 h. Activated cleaved caspase-3 immunohistochemical staining was used for detection of apoptosis. Cognitive functions were tested by pavlovian conditioned fear test. Social behavior was tested by social recognition and interaction tests. Results:Neonatal exposure to sevoflurane significantly increased the number of apoptotic cells in the brain immediately after anesthesia. It caused persistent learning deficits later in adulthood as evidenced by decreased freezing response in both contextual and cued fear conditioning. The social recognition test demonstrated that mice with neonatal exposure to sevoflurane did not develop social memory. Furthermore, these mice showed decreased interactions with a social target compared with controls in the social interaction test, indicating a social interaction deficit. The authors did not attribute these abnormalities in social behavior to impairments of general interest in novelty or olfactory sensation, because they did not detect significant differences in the test for novel inanimate object interaction or for olfaction. Conclusions:This study shows that exposure of neonatal mice to inhaled sevoflurane could cause not only learning deficits but also abnormal social behaviors resembling autism spectrum disorder.

Extracellular Signal-Regulated Kinase 2 (ERK2) Knockdown Mice Show Deficits in Long-Term Memory; ERK2 Has a Specific Function in Learning and Memory

The extracellular signal-regulated kinase (ERK) 1 and 2 are important signaling components implicated in learning and memory. These isoforms display a high degree of sequence homology and share a similar substrate profile. However, recent findings suggest that these isoforms may have distinct roles: whereas ERK1 seems to be not so important for associative learning, ERK2 might be critically involved in learning and memory. Thus, the individual role of ERK2 has received considerable attention, although it is yet to be understood. Here, we have generated a series of mice in which ERK2 expression decreased in an allele dose-dependent manner. Null ERK2 knock-out mice were embryonic lethal, and the heterozygous mice were anatomically impaired. To gain a better understanding of the influence of ERK2 on learning and memory, we also generated knockdown mice in which ERK2 expression was partially (20–40%) reduced. These mutant mice were viable and fertile with normal appearance. The mutant mice showed a deficit in long-term memory in classical fear conditioning, whereas short-term memory was normal. The mice also showed learning deficit in the water maze and the eight-arm radial maze. The ERK1 expression level of the knockdown mice was comparable with the wild-type control. Together, our results indicate a noncompensable role of ERK2-dependent signal transduction in learning and memory.

The amino-acid sequence of the nonspecific lipid transfer protein from germinated castor bean endosperms

Abstract The amino-acid sequence of the nonspecific lipid-transfer protein from germinated castor bean endosperms has been determined by automatic sequencing of Staphylococcus aureus proteinase and tryptic peptides. The protein has 92 residues and a molecular weight of 9313. The complete primary structure of this protein is: Val-Asp-Cys-Gly-Gln-Val-Asn-Ser-Ser-Leu10-Ala-Ser-Cys-Ile-Pro-Phe-Leu-Thr-Gly-Gly20-Val-Ala-Ser-Pro-Ser-Ala-Ser-Cys-Cys-Ala30-Gly-Val-Gln- Asn-Leu-Lys-Thr-Leu-Ala-Pro40-Thr-Ser-Ala-Asp-Arg-Arg-Ala-Ala-Cys-Glu50-Cys-Ile-Lys-Ala-Ala-Ala-Ala-Arg-Phe-Pro60-Thr-Ile-Lys-Gln-Asp-Ala- Ala-Ser-Ser-Leu70-Pro-Lys-Lys-Cys-Gly-Val-Asp-Ile-Asn-Ile80-Pro-Ile-Ser-Lys-Thr-Thr-Asn-Cys-Gln-Ala90-Ile-Asn. Sequence microheterogeneity was found at residues 42 and 50, suggesting the occurrence of two genes for this protein or the allelic variation of the same gene. 12 of 14 acidic and basic amino acids were located on the latter half of the sequence. The first 20 residues of this protein have a homology (45%) with the residues 2–21 of the lipid-transfer protein from spinach leaf.

Targeted DNA transfection into the mouse central nervous system using laser- induced stress waves

We investigated the feasibility of gene transfer into the mouse central nervous system (CNS) by applying nanosecond pulsed laser-induced stress waves (LISWs). Intraventricular or hippocampal injection of a reporter gene [enhanced green fluorescent protein (EGFP)] followed by application of LISWs showed this method to be efficient in the CNS of newborn and adult mice. Cells expressing EGFP reside at least 3.5 mm from the surface of the tissue, while no apparent damage was detected. Additionally, expression of EGFP was limited to the area that was exposed to LISWs. Using this method, the formulation of plasmid DNA by cationic transfer reagent polyethylenimine proved to be effective for improving transfer efficiency into the CNS.

Analysis of Extracellular Signal‐Regulated Kinase 2 Function in Neural Stem/Progenitor Cells via Nervous System‐Specific Gene Disruption

Extracellular signal‐regulated kinase 2 (ERK2) is involved in a variety of cell fate decisions during development, but its exact role in this process remains to be determined. To specifically focus on the role of ERK2 in the brain, and to avoid early lethalities, we used a conditional gene‐targeting approach to preferentially inactivate Erk2 in the embryonic mouse brain. The resulting mutant mice were viable and were relatively normal in overall appearance. However, the loss of Erk2 resulted in a diminished proliferation of neural stem cells in the embryonic ventricular zone (VZ), although the survival and differentiation of these cells was unaffected. The multipotent neural progenitor cells (NPCs) isolated from ERK2‐deficient brains also showed impaired proliferation, reduced self‐renewal ability, and increased apoptosis. By neurosphere differentiation analysis we further observed that lineage‐restricted glial progenitors were increased in ERK2‐deficient mice. The decline in the self‐renewal ability and multipotency of NPCs resulting from the loss of ERK2 was found to be caused at least in part by upregulation of the JAK‐STAT signaling pathway and reduced G1/S cell cycle progression. Furthermore, by global expression analysis we found that neural stem cell markers, including Tenascin C NR2E1 (Tlx), and Lgals1 (Galectin‐1), were significantly downregulated, whereas several glial lineage markers were upregulated in neurospheres derived from ERK2‐deficient mice. Our results thus suggest that ERK2 is required both for the proliferation of neural stem cells in the VZ during embryonic development and in the maintenance of NPC multipotency by suppressing the commitment of these cells to a glial lineage.

Dopamine D2 receptor stimulation promotes the proliferation of neural progenitor cells in adult mouse hippocampus.

We initially examined the effects of apomorphine in vitro using mouse embryonic and adult neural progenitor cells. The effects of apomorphine treatment led to dose-dependent increases in the number of embryonic and adult neural progenitor cells, and dopamine D2 receptor antagonist treatment significantly reduced the increases induced by apomorphine. Next, we investigated the effects of apomorphine in vivo in the adult mouse hippocampus. The effects of single-dose apomorphine administration led to an increase of approximately 30% in the number of bromodeoxyuridine-positive cells in the dentate gyrus. Moreover, the chronic apomorphine administration induced an increase in the number of bromodeoxyuridine-positive cells by about 30%. Thus, we suggest that the stimulation of dopamine D2 receptors increases the proliferation of neural progenitor cells both in vivo and in vitro.

ERK1 and ERK2 are required for radial glial maintenance and cortical lamination

ERK1/2 is involved in a variety of cellular processes during development, but the functions of these isoforms in brain development remain to be determined. Here, we generated double knockout (DKO) mice to study the individual and combined roles of ERK1 and ERK2 during cortical development. Mice deficient in Erk2, and more dramatically in the DKOs, displayed proliferation defects in late radial glial progenitors within the ventricular zone, and a severe disruption of lamination in the cerebral cortex. Immunohistochemical analyses revealed that late‐generated cortical neurons were misplaced and failed to migrate the upper cortical layers in DKO mice. Moreover, these mice displayed fewer radial glial fibers, which provide architectural guides for radially migrating neurons. These results suggest that extracellular signal‐regulated kinase signaling is essential for the expansion of the radial glial population and for the maintenance of radial glial scaffolding. Tangential migration of interneurons and oligodendrocytes from the ganglionic eminences (GE) to the dorsal cortex was more severely impaired in DKO mice than in mice deficient for Erk2 alone, because of reduced progenitor proliferation in the GE of the ventral telencephalon. These data demonstrate functional overlaps between ERK1 and ERK2 and indicate that extracellular signal‐regulated kinase signaling plays a crucial role in cortical development.

Tissue factor is associated with the nonbacterial thrombotic endocarditis induced by a hypobaric hypoxic environment in rats

Abstract High-altitude hypoxia causes a hypercoagulable state. In our previous study on the blood coagulation system in rats, nonbacterial thrombotic endocarditis (NBTE) developed after 4–12 weeks’ exposure to the equivalent of 5500 m in altitude. We hypothesized that TF (tissue factor)-producing cells in the cardiac valves might be induced by the hypobaric hypoxic environment (HHE) and then trigger NBTE. A total of 170 male Wistar rats were housed in a chamber at the equivalent of 5500 m altitude for 1–12 weeks. We measured TF activity in the plasma and studied morphological changes in the mitral valves using immunohistochemical and immunoelectrical methods for TF protein and in situ hybridization for TF mRNA. After 4 weeks or more of exposure to HHE, 28 of the 56 surviving rats had developed NBTE. After 4–8 weeks’ exposure to HHE, the plasma TF activity level was significantly higher than in control rats. There was a significant correlation between plasma TF activity and the incidence of NBTE. After 1 weeks’ exposure to HHE, immunoreactivity for TF protein was detected in foamy macrophages and stromal cells in the cardiac valves. In rats with NBTE, TF protein was present in foamy macrophages and spindle stromal cells and focally present in the extracellular matrix. TF mRNA was detected in some foamy macrophages within the thrombus, TF protein was localized to the rough endoplasmic reticulum and plasma membrane of many macrophages, some fibroblasts, and a few endocardial cells. TF is associated with the pathogenesis of the NBTE induced by exposure to HHE. The accumulation of TF-producing macrophages during exposure to HHE may be responsible for initiating thrombus formation.

Pulmonary blast injury in mice: a novel model for studying blast injury in the laboratory using laser-induced stress waves.

Primary blast injury is produced by shock waves. Blast injuries to lungs are extremely critical threats to survival, but their etiology is largely undefined. The majority of animal models for these injuries use explosive or complex experimental settings, limiting the laboratory study of blast injury. The aim of this study was to establish a small‐animal model for blast injuries, using laser‐induced stress waves (LISWs) with high controllability, high reproducibility, and easy experimental settings.

ERK2 dependent signaling contributes to wound healing after a partial-thickness burn.

Burn healing is a complex physiological process involving multiple cell activities, such as cell proliferation, migration and differentiation. Although extracellular signal-regulated kinases (ERK) have a pivotal role in regulating a variety of cellular responses, little is known about the individual functions of ERK isoform for healing in vivo. This study investigated the role of ERK2 in burn healing. To assess this, Erk2(+/-) mice generated by gene targeting were used. The resultant mice exhibited significant delay in re-epithelization of partial-thickness burns in the skin in comparison to wild-type. An in vitro proliferation assay revealed that keratinocytes from Erk2(+/-) mice grew significantly slower than those prepared from wild-type. These results highlight the importance of ERK2 in the process of burn healing.