Distribution, development and proliferation of interstitial cells of Cajal in murine colon: an immunohistochemical study from neonatal to adult life

This paper aimed at investigating the alterations in interstitial cells of Cajal (ICC) in the proximal, middle and distal colon of mice from 0-day to 56-day post-partum (P0–P56) by immunohistochemistry. The Kit⁺ ICC, which situated around myenteric nerve plexus (ICC–MY) were prominent at birth, meanwhile those cells within the smooth muscle layers (ICC–IM) and in the connective tissue beneath serosa (ICC–SS) began to appear. ICC–SM, which located at the submucosal border of circular muscle layer emerged at P6 in the proximal colon and subsequently in the distal colon at P8, and ICC in the oral side of colon revealed an earlier development in morphology and a higher density than that in the anal side. The density of ICC altered obviously during postnatal period, and the estimated total amount of ICC increased ~30 folds at P56 than that at P0. Some Kit⁺/Ki67⁺ and Kit⁺/BrdU⁺ cells were observed in ICC–MY, ICC–IM and ICC–SS, but not in ICC–SM from P0 to P24. Our result indicates a proximal to distal and transmural gradient development of ICC in the postnatal colon along with a dramatic increase of ICC cell number from neonatal to adult life, and an age-dependent proliferation of ICC is also involved.     

Detection of BrdU-label retaining cells in the lacrimal gland: implications for tissue repair

The purpose of the present study was to determine if the lacrimal gland contains 5-bromo-2′-deoxyuridine (BrdU)-label retaining cells and if they are involved in tissue repair. Animals were pulsed daily with BrdU injections for 7 consecutive days. After a chase period of 2, 4, or 12 weeks, the animals were sacrificed and the lacrimal glands were removed and processed for BrdU immunostaining. In another series of experiments, the lacrimal glands of 12-week chased animals were either left untreated or were injected with interleukin 1 (IL-1) to induce injury. Two and half days post-injection, the lacrimal glands were removed and processed for BrdU immunostaining. After 2 and 4 weeks of chase period, a substantial number of lacrimal gland cells were BrdU⁺ (11.98 ± 1.84 and 7.95 ± 1.83 BrdU⁺ cells/mm², respectively). After 12 weeks of chase, there was a 97% decline in the number of BrdU⁺ cells (0.38 ± 0.06 BrdU⁺ cells/mm²), suggesting that these BrdU-label retaining cells may represent slow-cycling adult stem/progenitor cells. In support of this hypothesis, the number of BrdU labeled cells increased over 7-fold during repair of the lacrimal gland (control: 0.41 ± 0.09 BrdU⁺ cells/mm²; injured: 2.91 ± 0.62 BrdU⁺ cells/mm²). Furthermore, during repair, among BrdU⁺ cells 58.2 ± 3.6 % were acinar cells, 26.4 ± 4.1% were myoepithelial cells, 0.4 ± 0.4% were ductal cells and 15.0 ± 3.0% were stromal cells. We conclude that the murine lacrimal gland contains BrdU-label retaining cells that are mobilized following injury to generate acinar, myoepithelial and ductal cells.     

Relation Among Neuronal Death,Cell Proliferation and Neuronal Differentiation in the Gerbil Main Olfactory Bulb after Transient Cerebral Ischemia

Neurogenesis occurs during the embryonic stage and throughout life. Brain injuries such as ischemic insults enhance cell proliferation in some areas of the brain. We examined proliferation of newly generated cells in each layer of the gerbil main olfactory bulb (MOB) after 5 min of transient cerebral ischemia using bromodeoxyuridine (BrdU) immunohistochemistry. Ischemia-related neuronal death in the MOB was not detected using Fluoro-Jade B histofluorescence and TUNEL staining. Many BrdU-positive (⁺) cells were found in the rostral migratory stream in control and ischemic MOBs. Significant increase of BrdU⁺ cells was observed in the granule cell layer (GCL) and glomerular layer (GL) from 15 days post-ischemia, and BrdU⁺ cells were very much higher than those of the control group 30 days post-ischemia. At this time point after ischemia/reperfusion, a few BrdU⁺ cells in the GL and GCL were co-localized with calretinin⁺ cells, and many BrdU⁺ cells expressed doublecortin, a marker of immature neurons. These results indicate that cell proliferation is increased in the GCL and GL without apparent neuronal loss from 15 days after transient cerebral ischemia in gerbils.     

Quantification and localization of erythropoietin-receptor-expressing cells in the liver of Xenopus laevis

Erythropoiesis occurs in the African clawed frog, Xenopus laevis and is mediated by erythropoietin (xlEPO), a primary regulator of this process. Previously, we have shown that the xlEPO receptor (xlEPOR), which is expressed by erythroid progenitors that respond to xlEPO, is found predominantly in the liver. The aim of the present study was to determine the dynamics of erythropoiesis in the livers of normal and anemic X. laevis by identifying the number and precise location of mature and immature erythrocytes. We quantified mature and immature erythrocyte numbers by o-dianisidine staining or immunohistochemistry and investigated the dynamics of erythropoiesis in normal, acute hemolytic and blood-loss states by in vivo cell proliferation assays with 5-bromo-2′-deoxyuridine (BrdU). We detected 0.12×10⁸ xlEPOR⁺ BrdU⁺ cells in the liver of the normal X. laevis at 24 h after BrdU injection. Frogs presenting with acute hemolytic anemia and pancytopenia show a 10-fold increase in the number of xlEPOR⁺/BrdU⁺ cells (approximately 1.30×10⁸ cells) in the liver. The xlEPOR⁺ cells are found predominantly on the inner wall of hepatic sinusoids. Hematopoietic progenitors that undergo slow cell cycling were also observed in the hepatic sinusoids. This study clarifies the rate of production of mature and immature erythrocytes per day in the liver of X. laevis and the way that these cell numbers change in response to anemia.     

Cerebellar stem cells do not produce neurons and astrocytes in adult mouse

Although previous studies implied that cerebellar stem cells exist in some adult mammals, little is known about whether these stem cells can produce new neurons and astrocytes. In this study by bromodeoxyuridine (BrdU) intraperitoneal (i.p.) injection, we found that there are abundant BrdU⁺ cells in adult mouse cerebellum, and their quantity and density decreases significantly over time. We also found cell proliferation rate is diversified in different cerebellar regions. Among these BrdU⁺ cells, very few are mash1⁺ or nestin⁺ stem cells, and the vast majority of cerebellar stem cells are quiescent. Data obtained by in vivo retrovirus injection indicate that stem cells do not produce neurons and astrocytes in adult mouse cerebellum. Instead, some cells labeled by retrovirus are Iba1⁺ microglia. These results indicate that very few stem cells exist in adult mouse cerebellum, and none of these stem cells contribute to neurogenesis and astrogenesis under physiological condition.     

Comparison of Neurogenesis in the Dentate Gyrus Between the Adult and Aged Gerbil Following Transient Global Cerebral Ischemia

In the present study, we compared differences in cell proliferation, neuroblast differentiation and neuronal maturation in the hippocampal dentate gyrus (DG) between the adult and aged gerbil induced by 5 min of transient global cerebral ischemia using Ki-67 and BrdU (markers for cell proliferation), doublecortin (DCX, a marker for neuroblast differentiation) and neuronal nuclei (NeuN, a marker for mature neuron). The number of Ki-67-immunoreactive (⁺) cells in the DG of both the groups peaked 7 days after ischemia/reperfusion (I/R). However, the number in the aged DG was 40.6 ± 1.8% of that in the adult DG. Thereafter, the number decreased with time. After ischemic damage, DCX immunoreactivity and its protein level in the adult and aged DG peaked at 10 and 15 days post-ischemia, respectively. However, DCX immunoreactivity and its protein levels in the aged DG were much lower than those in the adult. DCX immunoreactivity and its protein level in the aged DG were 11.1 ± 0.6% and 34.4 ± 2.1% of the adult DG, respectively. In addition, the number of Ki-67⁺ cells and DCX immunoreactivity in both groups were similar to those in the sham at 60 days postischemia. At 30 days post-ischemia, the number of BrdU⁺ cells and BrdU⁺/NeuN⁺ cells in the adult-group were much higher (281.2 ± 23.4% and 126.4 ± 7.4%, respectively) than the aged-group (35.6 ± 6.8% and 79.5 ± 6.1%, respectively). These results suggest that the ability of neurogenesis in the ischemic aged DG is much lower than that in the ischemic adult DG.     

Effects of long term nitrite therapy on functional recovery in experimental ischemia model

Our data have shown that nitrite therapy can rescue the ischemic brain when injected <3 h after cerebral ischemic-reperfusion (I/R) injury and its effects can be prolonged to 4.5 h in combination with memantine. We investigated whether or not long-term nitrite therapy is beneficial in ischemic brains. Sodium nitrite (1-100 μg/kg ip) or saline were administered to rats subjected to focal I/R injury for 7 days beginning 24 h after I/R. Behavioral tests for 5 weeks revealed better functional recovery in the high-dose nitrite group than the control group. Other nitrite groups with relatively low doses showed no functional benefits. Hemispheric atrophy was attenuated by approximately 30% in the high-dose nitrite group. High-dose nitrite therapy also reduced inflammatory cytokine levels and caspase activity in the subacute period, and increased BrdU⁺MAP2⁺ and BrdU⁺laminin⁺ cells, and vascular density in the 5-week ischemic brain. Long-term nitrite therapy, when initiated 24 h after I/R, corrected the subacute hostile environment, induced tissue and vascular regeneration, and improved functional recovery. Early and subsequent long term nitrite therapy may be effective in the management for ischemic stroke patients.     

Detection of reactive oxygen species in isolated, perfused lungs by electron spin resonance spectroscopy

Background

Interleukin (IL)-9 is a Th2-derived cytokine with pleiotropic biological effects, which recently has been proposed as a candidate gene for asthma and allergy. We aimed to evaluate the therapeutic effect of a neutralizing anti-IL-9 antibody in a mouse model of airway eosinophilic inflammation and compared any such effect with anti-IL-5 treatment.

Methods

OVA-sensitized Balb/c mice were intraperitoneally pretreated with a single dose (100 μg) of an anti-mouse IL-9 monoclonal antibody (clone D9302C12) or its vehicle. A third group was given 50 μg of a monoclonal anti-mouse IL-5 antibody (TRFK-5) or its vehicle. Animals were subsequently exposed to OVA on five days via airways. Newly produced eosinophils were labelled using 5-bromo-2'-deoxyuridine (BrdU). BrdU⁺ eosinophils and CD34⁺ cell numbers were examined by immunocytochemistry. After culture and stimulation with OVA or PMA+IC, intracellular staining of IL-9 in bone marrow cells from OVA-exposed animals was measured by Flow Cytometry. The Mann-Whitney U-test was used to determine significant differences between groups.

Results

Anti-IL-9 significantly reduced bone marrow eosinophilia, primarily by decrease of newly produced (BrdU⁺) and mature eosinophils. Anti-IL-9 treatment also reduced blood neutrophil counts, but did not affect BAL neutrophils. Anti-IL-5 was able to reduce eosinophil numbers in all tissue compartments, as well as BrdU⁺ eosinophils and CD34⁺ progenitor cells, and in all instances to a greater extent than anti-IL-9. Also, FACS analysis showed that IL-9 is over-expressed in bone marrow CD4⁺ cells after allergen exposure.

Conclusions

Our data shows that a single dose of a neutralizing IL-9 antibody is not sufficient to reduce allergen-induced influx of newly produced cells from bone marrow to airways. However, in response to allergen, bone marrow cells over-express IL-9. This data suggest that IL-9 may participate in the regulation of granulocytopoiesis in allergic inflammation.     

Cell proliferation and survival in the mating circuit of adult male hamsters: effects of testosterone and sexual behavior

The transient actions of gonadal steroids on the adult brain facilitate social behaviors, including reproduction. In male rodents, testosterone acts in the posterior medial amygdala (MeP) and medial preoptic area (MPOA) to promote mating. Adult neurogenesis occurs in both regions. The current study determined if testosterone and/or sexual behavior promote cell proliferation and survival in MeP and MPOA. Two experiments were conducted using the thymidine analog BrdU. First, gonad-intact and castrated male hamsters (n = 6/group) were compared 24 h or 7 weeks after BrdU. In MeP, testosterone-stimulated cell proliferation 24 h after BrdU (intact: 22.8 ± 3.9 cells/mm², castrate: 13.2 ± 1.4 cells/mm²). Testosterone did not promote cell proliferation in MPOA. Seven weeks after BrdU, cell survival was sparse in both regions (MeP: 2.5 ± 0.6 and MPOA: 1.7 ± 0.2 cells/mm²), and was not enhanced by testosterone. In Experiment 2, gonad-intact sexually-experienced animals were mated weekly to determine if regular neural activation enhances cell survival 7 weeks after BrdU in MeP and MPOA. Weekly mating failed to increase cell survival in MeP (8.1 ± 1.6 vs. 9.9 ± 3.2 cells/mm²) or MPOA (3.9 ± 0.7 vs. 3.4 ± 0.3 cells/mm²). Furthermore, mating at the time of BrdU injection did not stimulate cell proliferation in MeP (8.9 ± 1.7 vs. 8.1 ± 1.6 cells/mm²) or MPOA (3.6 ± 0.5 vs. 3.9 ± 0.7 cells/mm²). Taken together, our results demonstrate a limited capacity for neurogenesis in the mating circuitry. Specifically, cell proliferation in MeP and MPOA are differentially influenced by testosterone, and the birth and survival of new cells in either region are not enhanced by reproductive activity.     

The Selective Antagonism of P2X7 and P2Y1 Receptors Prevents Synaptic Failure and Affects Cell Proliferation Induced by Oxygen and Glucose Deprivation in Rat Dentate Gyrus

Purinergic P2X and P2Y receptors are broadly expressed on both neurons and glial cells in the central nervous system (CNS), including dentate gyrus (DG). The aim of this research was to determine the synaptic and proliferative response of the DG to severe oxygen and glucose deprivation (OGD) in acute rat hippocampal slices and to investigate the contribution of P2X₇ and P2Y₁ receptor antagonism to recovery of synaptic activity after OGD. Extracellular field excitatory post-synaptic potentials (fEPSPs) in granule cells of the DG were recorded from rat hippocampal slices. Nine-min OGD elicited an irreversible loss of fEPSP and was invariably followed by the appearance of anoxic depolarization (AD). Application of MRS2179 (selective antagonist of P2Y₁ receptor) and BBG (selective antagonist of P2X₇ receptor), before and during OGD, prevented AD appearance and allowed a significant recovery of neurotransmission after 9-min OGD. The effects of 9-min OGD on proliferation and maturation of cells localized in the subgranular zone (SGZ) of slices prepared from rats treated with 5-Bromo-2′-deoxyuridine (BrdU) were investigated. Slices were further incubated with an immature neuron marker, doublecortin (DCX). The number of BrdU⁺ cells in the SGZ was significantly decreased 6 hours after OGD. This effect was antagonized by BBG, but not by MRS2179. Twenty-four hours after 9-min OGD, the number of BrdU⁺ cells returned to control values and a significant increase of DCX immunofluorescence was observed. This phenomenon was still evident when BBG, but not MRS2179, was applied during OGD. Furthermore, the P2Y₁ antagonist reduced the number of BrdU⁺ cells at this time. The data demonstrate that P2X₇ and P2Y₁ activation contributes to early damage induced by OGD in the DG. At later stages after the insult, P2Y₁ receptors might play an additional and different role in promoting cell proliferation and maturation in the DG.     

Airway allergen exposure stimulates bone marrow eosinophilia partly via IL-9

Background

Interleukin (IL)-9 is a Th2-derived cytokine with pleiotropic biological effects, which recently has been proposed as a candidate gene for asthma and allergy. We aimed to evaluate the therapeutic effect of a neutralizing anti-IL-9 antibody in a mouse model of airway eosinophilic inflammation and compared any such effect with anti-IL-5 treatment.

Methods

OVA-sensitized Balb/c mice were intraperitoneally pretreated with a single dose (100 μg) of an anti-mouse IL-9 monoclonal antibody (clone D9302C12) or its vehicle. A third group was given 50 μg of a monoclonal anti-mouse IL-5 antibody (TRFK-5) or its vehicle. Animals were subsequently exposed to OVA on five days via airways. Newly produced eosinophils were labelled using 5-bromo-2''-deoxyuridine (BrdU). BrdU⁺ eosinophils and CD34⁺ cell numbers were examined by immunocytochemistry. After culture and stimulation with OVA or PMA+IC, intracellular staining of IL-9 in bone marrow cells from OVA-exposed animals was measured by Flow Cytometry. The Mann-Whitney U-test was used to determine significant differences between groups.

Results

Anti-IL-9 significantly reduced bone marrow eosinophilia, primarily by decrease of newly produced (BrdU⁺) and mature eosinophils. Anti-IL-9 treatment also reduced blood neutrophil counts, but did not affect BAL neutrophils. Anti-IL-5 was able to reduce eosinophil numbers in all tissue compartments, as well as BrdU⁺ eosinophils and CD34⁺ progenitor cells, and in all instances to a greater extent than anti-IL-9. Also, FACS analysis showed that IL-9 is over-expressed in bone marrow CD4⁺ cells after allergen exposure.

Conclusions

Our data shows that a single dose of a neutralizing IL-9 antibody is not sufficient to reduce allergen-induced influx of newly produced cells from bone marrow to airways. However, in response to allergen, bone marrow cells over-express IL-9. This data suggest that IL-9 may participate in the regulation of granulocytopoiesis in allergic inflammation.     

Yangyin Tongnao granules enhance neurogenesis in the peri-infarct area and upregulate brain-derived neurotrophic factor and vascular endothelial growth factor after focal cerebral ischemic infarction in rats

Yangyin Tongnao granules (YYTNG) have been extensively applied in the treatment of brain injury, mainly due to its antioxidant effects, inhibition of apoptosis, and enhancement of blood circulation. To analyze the effect of YYTNG on the recovery of neurological function and neurogenesis in the peri-infarct area after cerebral ischemic infarction in rats and to elucidate its role in the neuroprotective mechanism of stroke, Sprague–Dawley (SD) rats were subjected to middle cerebral artery occlusion (MCAO) for 90 min followed by reperfusion. Rats were randomly divided into five groups: sham, MCAO, and YYTNG-treated rats given doses of 0.83, 1.65, or 3.3 g kg^?1 day^?1. The YYTNG-treated groups (1.65 and 3.3 g kg^?1 day^?1) showed higher neurological scores and a lower infarct volume than the MCAO group on day 3 after MCAO. Furthermore, the YYTNG-treated groups (0.83, 1.65, and 3.3 g kg^?1 day^?1) showed higher neurological scores on day 7 after MCAO. The number of BrdU⁺/nestin⁺, BrdU⁺/NeuN⁺, and BrdU⁺/GFAP⁺ cells in the peri-infarct area 7 days after MCAO was significantly increased in the YYTNG-treated groups in a dose-dependent manner. The protein expression levels of brain-derived neurotrophic factor (BDNF) and vascular endothelial growth factor (VEGF) were significantly higher in all three YYTNG-treated groups than in the MCAO group. Based on these results, administration of YYTNG post ischemia could ameliorate neurological function deficits in rats with MCAO. The therapeutic effect of YYTNG may be due to the promotion of neurogenesis in the peri-infarct area and the upregulation of neuroprotective factors BDNF and VEGF in MCAO rats.

     

Infiltrated Cells in Experimental Allergic Encephalomyelitis by Additional Intracerebral Injection in Myelin-Basic-Protein-SensitizedB6 Mice

We previously reported that murine experimental allergic encephalomyelitis can be induced by an additional intraperitoneal and intracerebral (i.c.) restimulation in resistant B6 mice after standard immunization with myelin antigens in complete Freund's adjuvant andBordetella pertussis coadjuvant. Neutrophils infiltrated into perivascular spaces at 12 h, followed by mononuclear cells 24 h after i.c. injection. In this study, we report that the i.c. injection induced the expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). The kinetic expression of ICAM-1 or VCAM-1 on brain endothelial cells paralleled the infiltration of neutrophils and mononuclear cells, respectively. The infiltrated lymphocytes also expressed very late antigen-4 (VLA-4) molecules. The microvascular endothelial cells were positive for VCAM-1, whereas the surrounding mononuclear cells were VLA-4 positive. Furthermore, we found a unique subpopulation of cells with characteristics of CD4-CD8-V8⁺ markers. The kinetic studies of this population showed that these cells were transiently depleted from 12 to 24 h after i.c. challenge (before the development of clinical symptoms) in cervical lymph nodes. These CD4-CD8-V8⁺ cells can be expanded by in vitro culture with myelin basic protein or IL-2. No significant changes of CD4⁺/CD8⁺ cells were noted. CD4⁺CD8^–CD3⁺ cells were also found in brain by double histochemical stains and were the major infiltrating cells at 24 or 48 h after i.c. challenge.     

Mast cells in a murine lung ischemia-reperfusion model of primary graft dysfunction

Primary graft dysfunction (PGD), as characterized by pulmonary infiltrates and high oxygen requirements shortly after reperfusion, is the major cause of early morbidity and mortality after lung transplantation. Donor, recipient and allograft-handling factors are thought to contribute, although new insights regarding pathogenesis are needed to guide approaches to prevention and therapy. Mast cells have been implicated in ischemic tissue injury in other model systems and in allograft rejection, leading to the hypothesis that mast cell degranulation contributes to lung injury following reperfusion injury.We tested this hypothesis in a mouse model of PGD involving reversible disruption of blood flow to one lung. Metrics of injury included albumin permeability, plasma extravasation, lung histopathology, and mast cell degranulation. Responses were assessed in wild-type (Kit^+/+) and mast cell-deficient (Kit^W-sh/W-sh) mice. Because mouse lungs have few mast cells compared with human lungs, we also tested responses in mice with lung mastocytosis generated by injecting bone marrow-derived cultured mast cells (BMCMC).We found that ischemic lung responses of mast cell-deficient Kit^W-sh/W-sh mice did not differ from those of Kit^+/+ mice, even after priming for injury using LPS. Degranulated mast cells were more abundant in ischemic than in non-ischemic BMCMC-injected Kit^W-sh/W-sh lungs. However, lung injury in BMCMC-injected Kit^W-sh/W-sh and Kit^+/+ mice did not differ in globally mast cell-deficient, uninjected Kit^W-sh/W-sh mice or in wild-type Kit^+/+ mice relatively deficient in lung mast cells.These findings predict that mast cells, although activated in lungs injured by ischemia and reperfusion, are not necessary for the development of PGD.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-014-0095-0) contains supplementary material, which is available to authorized users.     

Hu neuronal proteins are expressed in proliferating neurogenic cells

We have utilized immunochemical techniques to investigate the developmental expression of the Hu proteins, a neuron-specific family of RNA binding proteins in vertebrates. Previous work suggests that these proteins may play an important role in neuronal development and maintenance. For the present study, we developed a monoclonal antibody (MAb 16A11) that binds specifically to an epitope present in gene products of all known Hu genes, including HuD, HuC, and Hel-N1. Using brief pulses (1–2 h) of the DNA precursor analog bromodeoxyruridine (BrdU) in conjunction with MAb 16A11, we observed Hu⁺/BrU⁺ cells in nascent sensory and sympathetic ganglia in vivo, and in populations of cultured neural crest cells. In addition, a few Hu⁺ cells were ambiguously BrdU⁺ in the neural tube. We conclude that Hu+ cells first appear in avian neurogenic populations immediately before neuronal birthdays in the peripheral nervous system, and at the time of withdrawal from the mitotic cycle in the central nervous system. Consistent with these conclusions, we have also observed neural crest-derived cells that are both Hu+ and in metaphase of the cell cycle. We suggest that Hu proteins function early in neurogenic differentiation. 1994 John Wiley & Sons, Inc.     

Cortical Endogenic Neural Regeneration of Adult Rat after Traumatic Brain Injury

Focal and diffuse neuronal loss happened after traumatic brain injury (TBI). With little in the way of effective repair, recent interest has focused on endogenic neural progenitor cells (NPCs) as a potential method for regeneration. Whether endogenic neural regeneration happened in the cortex of adult rat after TBI remains to be determined. In this study, rats were divided into a sham group and a TBI group, and the rat model of medium TBI was induced by controlled cortical impact. Rats were injected with BrdU at 1 to 7 days post-injury (dpi) to allow identification of differentiated cells and sacrificed at 1, 3, 7, 14 and 28 dpi for immunofluorescence. Results showed nestin⁺/sox-2⁺ NPCs and GFAP⁺/sox-2⁺ radial glial (RG)-like cells emerged in peri-injured cortex at 1, 3, 7, 14 dpi and peaked at 3 dpi. The number of GFAP⁺/sox-2⁺ cells was less than that of nestin⁺/sox-2⁺ cells. Nestin⁺/sox-2⁺ cells from posterior periventricle (pPV) immigrated into peri-injured cortex through corpus callosum (CC) were found. DCX⁺/BrdU⁺ newborn immature neurons in peri-injured cortex were found only at 3, 7, 14 dpi. A few MAP-2⁺/BrdU⁺ newborn neurons in peri-injured cortex were found only at 7 and 14 dpi. NeuN⁺/BrdU⁺ mature neurons were not found in peri-injured cortex at 1, 3, 7, 14 and 28 dpi. While GFAP⁺/BrdU⁺ astrocytes emerged in peri-injured cortex at 1, 3, 7, 14, 28 dpi and peaked at 7 dpi then kept in a stable state. In the corresponding time point, the percentage of GFAP⁺/BrdU⁺ astrocytes in BrdU⁺ cells was more than that of NPCs or newborn neurons. No CNP⁺/BrdU⁺ oligodendrocytes were found in peri-injured cortex. These findings suggest that NPCs from pPV and reactive RG–like cells emerge in peri-injured cortex of adult rats after TBI. It can differentiate into immature neurons and astrocytes, but the former fail to grow up to mature neurons.     

Functional restoration of acoustic units and adult‐generated neurons after hypothalamic lesion

The hypothalamus of the adult ring dove contains acoustic units that respond to species‐specific coo vocalization. Loss of nest coo leads to unsuccessful breeding. However, the recovery of nest coo in some doves suggests that these units are capable of self‐renewal. We have previously shown that lesioning the hypothalamus generates the addition of new neurons at the lesioned area. In this study, we sought to determine whether lesion‐induced new neurons are involved in the recovery of coo‐responsive units. We systematically recorded electrical activity in the ventromedial nucleus (VMN) of the hypothalamus, before and after lesion, for varying periods up to 3 months. Recordings were made when the birds were at rest (spontaneous discharge) and when the birds were exposed to acoustic stimulations (evoked discharge). Concurrently, the lesioned area was monitored for changes in cell types by using bromodeoxyuridine (BrdU) to label newly divided cells and NeuN to identify mature neurons. For 1 month after lesion, there was no sign of electrical activity, and only BrdU‐labeled cells were present. When the first electrical activity occurred, it displayed abnormal spontaneous bursting patterns. The mature discharge patterns (both spontaneous and evoked) occurred after detection of BrdU⁺/NeuN⁺ double‐labeled cells 2–3 months postlesion and were similar to those found in intact and sham‐lesioned birds. Double‐labeled cells bore morphologic characteristics of a neuron and were confirmed with z‐stack analysis using confocal laser scanning microscopy. Moreover, double‐labeled cells were not stained for glial fibrillary acidic protein (GFAP), suggesting that they were neurons. The number of coo‐responsive units was significantly correlated with that of BrdU⁺/NeuN⁺ cells. Furthermore, the marker for recording sites revealed that coo‐responsive units were colocalized with BrdU⁺/NeuN⁺ cells. Taken together, the evidence strongly suggests that lesion‐induced addition of new neurons promotes the functional recovery of the adult hypothalamus. © 2004 Wiley Periodicals, Inc. J Neurobiol 60: 197–213, 2004     

Effect of vitrification of in vitro matured prepubertal goat oocytes on embryo development after parthenogenic activation and intracytoplasmic sperm injection

This work studies the effect of vitrification of in vitro matured (IVM) prepubertal goat oocytes on: 1) oocyte damage assessed by reactive oxygen species (ROS) level and apoptosis and 2) embryo development after Intracytoplasmic sperm injection (ICSI) and Parthenogenic Activation (PA). Oocytes were IVM in supplemented TCM-199 for 22–24 h. Control group oocytes matured during 24 h were directly used for the analysis after IVM. Vitrified/warmed IVM-oocytes were vitrified after 22 h of IVM in 15% ethylene glycol (EG), 15% dimethyl sulfoxide (Me₂SO) and 0.5 M sucrose and after subjected to warming procedure. Oocyte ROS level was measured by staining denuded IVM-oocytes with 10 μM 2′7′ dichlorodihydrofluorescein diacetate. Apoptosis was analyzed by Annexin V (AV) Apoptosis Detection kit and Propidium iodide (PI) signal and oocytes were classified as: Live (AV⁻ PI⁻), early apoptotic (AV⁺ PI⁻), dead non-apoptotic (AV⁻ PI⁺) and necrotic (AV⁺ PI⁺). Developmental competence of vitrified/warmed oocytes was assessed by PA (5 min in 5 μM Ionomycin plus 4 h in 2 mM 6-Dimethylaminopurine), and by ICSI fertilization. Presumptive zygotes were in vitro cultured for 8 days in commercial media BO-IVC. Vitrified/warmed oocytes showed higher ROS levels (P < 0.0001), lower live oocytes (44 vs. 66%; P: 0.0025) and higher dead non-apoptotic oocytes (33 vs. 13% P: 0.023) compared to control. No differences were found on normal zygote formation (2 PN) (32 vs. 25%) or blastocyst development (0 vs. 4%) after ICSI fertilization. However, after PA, significant differences were found in cleavage rate (59 vs.78%; P < 0.0343) and blastocyst formation (1 vs. 25%; P < 0.0001). In conclusion, vitrification reduced oocyte competence by increasing dead oocytes and ROS levels.     

Tanshinone I Enhances Neurogenesis in the Mouse Hippocampal Dentate Gyrus via Increasing Wnt-3, Phosphorylated Glycogen Synthase Kinase-3β and β-Catenin Immunoreactivities

Tanshinone I (TsI), a lipophilic diterpene extracted from Danshan (Radix Salvia miltiorrhizae), exerts neuroprotection in cerebrovascular diseases including transient ischemic attack. In this study, we examined effects of TsI on cell proliferation and neuronal differentiation in the subgranular zone (SGZ) of the mouse dentate gyrus (DG) using Ki-67, BrdU and doublecortin (DCX) immunohistochemistry. Mice were treated with 1 and 2 mg/kg TsI for 28 days. In the 1 mg/kg TsI-treated-group, distribution patterns of BrdU, Ki-67 and DCX positive (⁺) cells in the SGZ were similar to those in the vehicle-treated-group. However, in the 2 mg/kg TsI-treated-group, double labeled BrdU⁺/NeuN⁺ cells, which are mature neurons, as well as Ki-67⁺, DCX⁺ and BrdU⁺ cells were significantly increased compared with those in the vehicle-treated-group. On the other hand, immunoreactivities and protein levels of Wnt-3, β-catenin and serine-9-glycogen synthase kinase-3β (p-GSK-3β), which are related with morphogenesis, were significantly increased in the granule cell layer of the DG only in the 2 mg/kg TsI-treated-group. Therefore, these findings indicate that TsI can promote neurogenesis in the mouse DG and that the neurogenesis is related with increases of Wnt-3, p-GSK-3β and β-catenin immunoreactivities.