Doublecortin Research Articles

Constitutive Neurogenesis and Neuronal Plasticity in the Adult Cerebellum and Brainstem of Rainbow Trout, Oncorhynchus mykiss.

The central nervous system of Pacific salmon retains signs of embryonic structure throughout life and a large number of neuroepithelial neural stem cells (NSCs) in the proliferative areas of the brain, in particular. However, the adult nervous system and neurogenesis studies on rainbow trout, Oncorhynchus mykiss, are limited. Here, we studied the localization of glutamine synthetase (GS), vimentin (Vim), and nestin (Nes), as well as the neurons formed in the postembryonic period, labeled with doublecortin (DC), under conditions of homeostatic growth in adult cerebellum and brainstem of Oncorhynchus mykiss using immunohistochemical methods and Western Immunoblotting. We observed that the distribution of vimentin (Vim), nestin (Nes), and glutamine synthetase (GS), which are found in the aNSPCs of both embryonic types (neuroepithelial cells) and in the adult type (radial glia) in the cerebellum and the brainstem of trout, has certain features. Populations of the adult neural stem/progenitor cells (aNSPCs) expressing GS, Vim, and Nes have different morphologies, localizations, and patterns of cluster formation in the trout cerebellum and brainstem, which indicates the morphological and, obviously, functional heterogeneity of these cells. Immunolabeling of PCNA revealed areas in the cerebellum and brainstem of rainbow trout containing proliferating cells which coincide with areas expressing Vim, Nes, and GS. Double immunolabeling revealed the PCNA/GS PCNA/Vim coexpression patterns in the neuroepithelial-type cells in the PVZ of the brainstem. PCNA/GS coexpression in the RG was detected in the submarginal zone of the brainstem. The results of immunohistochemical study of the DC distribution in the cerebellum and brainstem of trout have showed a high level of expression of this marker in various cell populations. This may indicate: (i) high production of the adult-born neurons in the cerebellum and brainstem of adult trout, (ii) high plasticity of neurons in the cerebellum and brainstem of trout. We assume that the source of new cells in the trout brain, along with PVZ and SMZ, containing proliferating cells, may be local neurogenic niches containing the PCNA-positive and silent (PCNA-negative), but expressing NSC markers, cells. The identification of cells expressing DC, Vim, and Nes in the IX-X cranial nerve nuclei of trout was carried out.

Effect of 2-Week Naringin Supplementation on Neurogenesis and BDNF Levels in Ischemia–Reperfusion Model of Rats

Background: Ischemic stroke is the leading cause of mortality and disability worldwide with more than half of survivors living with serious neurological sequelae; thus, it has recently attracted a lot of attention in the field of medical study. Purpose: The aim of this study was to determine the effect of naringin supplementation on neurogenesis and brain-derived neurotrophic factor (BDNF) levels in the brain in experimental brain ischemia–reperfusion. Study design: The research was carried out on 40 male Wistar-type rats (10–12 weeks old) obtained from the Experimental Animals Research and Application Center of Selçuk University. Experimental groups were as follows: (1) Control group, (2) Sham group, (3) Brain ischemia–reperfusion group, (4) Brain ischemia–reperfusion + vehicle group (administered for 14 days), and (5) Brain ischemia–reperfusion + Naringin group (100 mg/kg/day administered for 14 days). Methods: In the ischemia–reperfusion groups, global ischemia was performed in the brain by ligation of the right and left carotid arteries for 30 min. Naringin was administered to experimental animals by intragastric route for 14 days following reperfusion. The training phase of the rotarod test was started 4 days before ischemia–reperfusion, and the test phase together with neurological scoring was performed the day before and 1, 7, and 14 days after the operation. At the end of the experiment, animals were sacrificed, and then hippocampus and frontal cortex tissues were taken from the brain. Double cortin marker (DCX), neuronal nuclear antigen marker (NeuN), and BDNF were evaluated in hippocampus and frontal cortex tissues by Real-Time qPCR analysis and immunohistochemistry methods. Results: While ischemia–reperfusion increased the neurological score values, DCX, NeuN, and BDNF levels decreased significantly after ischemia in the hippocampus and frontal cortex tissues. However, naringin supplementation restored the deterioration to a certain extent. Conclusion: The results of the study show that 2 weeks of naringin supplementation may have protective effects on impaired neurogenesis and BDNF levels after brain ischemia and reperfusion in rats.

A181 INFECTION WITH THE RAT TAPEWORM HYMENOLEPIS DIMINUTA REVEALS AN INTERLEUKIN-4 INDEPENDENT TUFT CELL ASSOCIATED WITH PEYER’S PATCHES

BackgroundInfection with the rat tapeworm Hymenolepis diminuta elicits a T helper 2 (Th2) imunity and suppresses dinitrobenzene sulphonic-acid-induced colitis in mice. However, the signaling cascade in an immunocompetent host that recognizes and mobilizies early immune events to expel the worm is poorly understood. To fully understand how helminth-infection can ameliorate concomitant disease, it is important to elucidate key cells/mediators in the detection of the worm and early events in the local anti-worm response. Here, we assess the chemosensory epithelial tuft cell and Peyer’s patches (PP) as the primary inductive sites of mucosal immunity in H. diminuta-infected mice.AimsTo assess the role of tuft cells in PP development and worm expulsion following infection of H. diminuta.MethodsBALB/c, BALB/c Il-4receptor-a-/-, C57Bl/6 and C57Bl/6 pou2f3-/- (transcription factor critial for tuft cell development) mice were infected with 5 cysticercoids of H. diminuta and necropsied 5-, 8- and 11-days post-infection (dpi); time-matched non-infected mice served as controls. PP number and size were counted and measured. Enteric tuft cells were assessed by immunostaining for the canonical marker double cortin like kinase (dclk)-1, and enumerated in PP-associated epithelium and villous epithelium distant from PP.ResultsDclkl+ tuft cells were sparse throughout villus epithelium of control mice, but were notably aggregrated over PPs. Infection with H. diminuta resulted in more detectable PPs by visual inspection, but did not result in a statistically significant increase in either size or number of PP. Infected mice showed increased numbers of dclk1+ tuft cells in villus epithelium and PP-associated epithelium (n=3–6). Tuft cells were absent in pou2f3-/- mice, that showed normal size and number of PPs ± infection with H. diminuta. Unexpectedly, analysis of small intestine from il4ra-/- mice revealed dclk1+ tuft cells in association with PPs; perhaps a unique sub-type of this sentinel cell.ConclusionsAnalysis of tuft cells after infection with H. diminuta revealed that these cells aggregate around PPs under homeostatic conditions, and future studies will explore if this distrirbution is important for ‘M’ cell development and/or PP immune reactivity. Intrigingly, an il-4ra-independent tuft cell subtype was observed that awaits fuller characterization. Understanding the tuft cell may reveal novel aspects of development of mucosal immunity relevant to combating helminth-infection and perhaps autoinflammatory enteric disease.Funding AgenciesNSERC

Molecular Markers of Adult Neurogenesis in the Telencephalon and Tectum of Rainbow Trout, Oncorhynchus mykiss.

In the brain of teleost fish, radial glial cells are the major type of astroglial cells. To answer the question as to how radial glia structures adapt to the continuous growth of the brain, which is characteristic of salmonids, it is necessary to study various types of cells (neuronal precursors, astroglial cells, and cells in a state of neuronal differentiation) in the major integrative centers of the salmon brain (telencephalon and tectum opticum), using rainbow trout, Oncorhynchus mykiss, as a model. A study of the distribution of several molecular markers in the telencephalon and tectum with the identification of neural stem/progenitor cells, neuroblasts, and radial glia was carried out on juvenile (three-year-old) O. mykiss. The presence of all of these cell types provides specific conditions for the adult neurogenesis processes in the trout telencephalon and tectum. The distribution of glutamine synthetase, a molecular marker of neural stem cells, in the trout telencephalon revealed a large population of radial glia (RG) corresponding to adult-type neural stem cells (NSCs). RG dominated the pallial region of the telencephalon, while, in the subpallial region, RG was found in the lateral and ventral zones. In the optic tectum, RG fibers were widespread and localized both in the marginal layer and in the periventricular gray layer. Doublecortin (DC) immunolabeling revealed a large population of neuroblasts formed in the postembryonic period, which is indicative of intense adult neurogenesis in the trout brain. The pallial and subpallial regions of the telencephalon contained numerous DC+ cells and their clusters. In the tectum, DC+ cells were found not only in the stratum griseum periventriculare (SGP) and longitudinal torus (TL) containing proliferating cells, but also in the layers containing differentiated neurons: the central gray layer, the periventricular gray and white layers, and the superficial white layer. A study of the localization patterns of vimentin and nestin in the trout telencephalon and tectum showed the presence of neuroepithelial neural stem cells (eNSCs) and ependymoglial cells in the periventricular matrix zones of the brain. The presence of vimentin and nestin in the functionally heterogeneous cell types of adult trout indicates new functional properties of these proteins and their heterogeneous involvement in intracellular motility and adult neurogenesis. Investigation into the later stages of neuronal development in various regions of the fish brain can substantially elucidate the major mechanisms of adult neurogenesis, but it can also contribute to understanding the patterns of formation of certain brain regions and the involvement of RG in the construction of the definite brain structure.

Grape seed extract effects on hippocampal neurogenesis, synaptogenesis and dark neurons production in old mice. Can this extract improve learning and memory in aged animals?

ABSTRACT Background During the elderly, hippocampal neurogenesis and synaptogenesis reduce and dark neurons (DNs) increase, leading to cognitive impairment. It is believed that natural products can protect the neural cells and system by protecting from damages or promoting regeneration. Therefore, the effects of grape seed extract (GSE) on the hippocampus of aged mice were investigated in this study. Methods twelve old mice were divided into two groups of control and GSE. Animals in the GSE group received 300 mg/kg of GSE for eight weeks via gavage. At the end of treatment, cognition performance was evaluated by Morris water maze (MWM) and passive avoidance tests. Hippocampal neurogenesis, synaptogenesis and DNs production were evaluated with immunohistochemistry and histological evaluations on 5-micron coronal tissue sections. Results The hippocampal mean number of double cortin positive cells (DCX+) per unit area, as well as synaptophysin expression in the GSE group, were significantly higher than the control group (p < 0.01). The frequency of DNs in the GSE group was lower than the control group (p < 0.05). Behavioral tests showed that GSE improves memory and learning performance. Conclusion Consuming GSE in the elderly can potentially alleviate the age-related reduction of hippocampal neurogenesis and synaptogenesis. It is also able to decrease hippocampal DNs production and increase memory and learning.

Pseudo‐repeats in doublecortin make distinct mechanistic contributions to microtubule regulation

Doublecortin (DCX) is a neuronal microtubule‐associated protein (MAP) indispensable for brain development. Its flexibly linked doublecortin (DC) domains—NDC and CDC—mediate microtubule (MT) nucleation and stabilization, but it is unclear how. Using high‐resolution time‐resolved cryo‐EM, we mapped NDC and CDC interactions with tubulin at different MT polymerization stages and studied their functional effects on MT dynamics using TIRF microscopy. Although coupled, each DC repeat within DCX appears to have a distinct role in MT nucleation and stabilization: CDC is a conformationally plastic module that appears to facilitate MT nucleation and stabilize tubulin–tubulin contacts in the nascent MT lattice, while NDC appears to be favored along the mature lattice, providing MT stabilization. Our structures of MT‐bound DC domains also explain in unprecedented detail the DCX mutation‐related brain defects observed in the clinic. This modular composition of DCX reflects a common design principle among MAPs where pseudo‐repeats of tubulin/MT binding elements chaperone or stabilize distinct conformational transitions to regulate distinct stages of MT dynamic instability.

Long non-coding RNA NEAT1 regulates Hodgkin's lymphoma cell proliferation and invasion via miR-448 mediated regulation of DCLK1.

To explore whether long non-coding RNA nuclear enriched abundant transcript 1 (lncRNA NEAT1) could regulate Hodgkin's lymphoma (HL) cell proliferation and invasion through miR-448, which could target doublecortin like kinase 1 (DCLK1) and mediate DCLK1 expression. Expressions of NEAT1, miR-448 and DCLK1 were evaluated by qRT-PCR or Western blot assay. Cell Counting Kit-8 (CCK-8) and transwell assay were utilized to detect cell proliferation and invasion capability in L428 cells respectively. The target relationship between NEAT1, miR-448 and DCLK1 was confirmed by Luciferase reporter assay. QRT-PCR results showed that NEAT1 expressed higher in HL tissues and cell lines than that in controls. In vitro experiments, NEAT1 downregulation could decrease cell proliferation and invasion capability in L428 cells. NEAT1 directly interacted with miR-448 and negatively regulated it. Moreover, DCLK1 was confirmed as a target of miR-448. DCLK1 expression was increased in L428 cells and positively regulated by NEAT1. NEAT1 overexpression upregulated the protein level of DCLK1 in L428 cells according to Western blot analysis. Additionally, DCLK1 overexpression could reverse the suppression on cell proliferation and invasion capability induced by NEAT1 knockdown or miR-448 overexpression. NEAT1 might be contributed to HL progression by promoting cell proliferation and invasion capability via miR-448 mediated DCLK1 expression.

Doublecortin like Kinase-1 is Overexpressed in Breast Cancer Tissues and Correlated with Epithelial-mesenchymal Transition Markers

Background: Much of the current literature support, the idea that epithelial to mesenchymal transition (EMT) is the key mechanism by which tumor cells gain invasive and metastatic ability, as EMT enables separation of individual cells from the primary tumor mass as well as promote migration. After undergoing EMT, thereby enabling access to hematogenous or lymphatic routes of dissemination, tumor cells can extravasate into secondary organs and establish micro-metastases. Objective: The main target of this work was to study the relation between expression of Doublecortin-like kinase-1 (DCLK-1) and epithelial to mesenchymal transition markers (E-cadherin, vimentin and transforming growth factor-beta (TGF-β)) in breast cancer patients and assess its role in cancer prognosis. Materials and Methods: This study included 60 breast cancer patients and 40 healthy females as control group. Tumor tissues and adjacent normal breast tissues were collected from patients with breast cancer. A single venous blood sample was collected concurrently from breast cancer patients (Before surgery) and from the control group. Tissue expression of DCLK-1, E-cadherin and vimentin were evaluated in breast cancer tissues and normal breast tissues by real-time reverse transcription polymerase chain reaction (RT-PCR). Serum level of TGF-β was assayed by enzyme linked-immunossorbant assay. Results: According to the results of the present study DCLK-1 is highly expressed in breast cancer tissues compared to normal breast tissues. Overexpression of DCLK-1 was significantly correlated with higher tissues Vimentin expression, increased serum TGF-β and lower tissues E-cadherin expression. Kaplan-Meier survival curves for breast cancer patients revealed that, patients with elevated tissue DCLK-1 and Vimentin expression and higher serum TGF-β were significantly associated with poor prognosis in primary breast cancer patients. However, patients with lower tissue E-Cadherin expression had shorter disease free survival time than patients with higher levels. Conclusion: DCLK-1 was significantly increased in breast cancer tissues in comparison to normal breast tissues and its overexpression was significantly correlated with EMT markers and poor prognosis in breast cancer patients. Further prospective studies using greater numbers of patients are required to confirm our findings.

Effects of total saponins of panax notoginseng on depressive behavior and hippocampal nerve regeneration in rats with following global cerebral ischemia depression

Objective To explore the effect of the total saponins of panax notoginseng (TSPN) on depression-like behavior following global cerebral ischemia depression in rats and its mechanism. Methods Using four-vessel occlusion method to build the global cerebral ischemia model, then the cerebral ischemia rats were given solitary breeding with chronic unpredictable mild stress(CUMS) to prepare depression model.Seventy rats were divided into sham group (n=10), model group (n=20), PSD group (n=20) and TSPN group (n=20). The rats in the TSPN group were administered TSPN intraperitoneally 30 min post-brain ischemia.The dose of TSPN (75mg/kg) was suspended in 0.9% saline 10g/L, once per day for 30 days after reperfusion.While rats in the vehicle group and PSD group was treated with equal volume of 0.9% saline, one injection per day until the rats were sacrificed at 30 days after brain ischemia.The BrdU, doublecortin (DCX) expression in the hippocampus was assessed by immunohistochemistry. Results In comparison with the model group, the sucrose preference percentage in the PSD group was significantly lower ((46.2±9.2)%, (61.2±7.6)%; t=3.18, P<0.05), then the PSD rats were administered TSPN intraperitoneally, the sucrose preference percentage increased significantly ((62.4±3.4)%, (46.2±9.2)%; t=3.43, P<0.05). During the forced swimming test, the immobility time of PSD group was significantly increased compared with the model group ((119.4±9.7)s, (88.0±15.6)s ; t=4.30, P<0.01), while after PSD rats administering TSPN intraperitoneally, the immobility time was shorten remarkably ((97.4±6.7)s, (119.4±9.7)s; t=3.01, P<0.05). Compared with the Model group(BrdU+ : (12.6±2.2)/mm2, DCX+ : (38.6±4.2)/mm2), the number of BrdU+ and DCX+ cells in the SGZ of hippocampus in PSD group decreased (BrdU+ : (8.8±1.5)/mm2, DCX+ : (27.2±2.8)/mm2; t=3.25, 4.29, both P<0.01). And compared with PSD group, the number of BrdU+ and DCX+ cells in the SGZ of hippocampus in TSPN group increased significantly (BrdU+ : (14.8±2.8)/mm2, DCX+ : (37.0±3.3)/mm2; t=4.68, 3.69, both P<0.05). Conclusion TSPN can improve the depression-like behavior of rats following global cerebral ischemia, which may be related with promoting hippocampal nerve regeneration. Key words: Total saponins of panax notoginseng; Hippocampus; Nerve regeneration; Following global cerebral ischemia depression; Rats

Effect of arsenic exposure on learning and memory in rat models and its underlying mechanisms

Objective To explore the effects of arsenic exposure on learning and memory and its potential mechanism in rats. Methods Water-based arsenic-exposed rat models were established on 4-10 postnatal days.The experimental animals were divided into 4 groups (10-12 cases in each group): the control group, the 15 μg/L As2O3 water group, the 30 μg/L As2O3 water group, and the 45 μg/L As2O3 water group.Cognitive functions were examined with the Morris water maze, exploratory behavior was detected by the exploratory behavior test.The hippocampus of pups from each experimental group was sectioned at various time points after arsenic exposure.The morphologies and neurogenesis of the neurons in the hippocampus CA1-CA3 region and dentate gyrus (DG) were observed by hematoxylin-eosin staining, Nissl staining, and doublecortin (DCX) immunostaining at different time points after arsenic exposure. Results Compared with the normal control group, the escape latency of the rats in the arsenic-exposed group was prolonged.The average escape latency of the rats in the normal control group, 15 μg/L As2O3 group, 30 μg/L As2O3 group and 45 μg/L As2O3 group were (17.00±9.53) s, (35.89±19.81) s, (26.60±18.84) s, and (33.79±18.08) s, respectively, and the difference among 4 groups was statistically significant (F=3.591, P<0.05), and the residence time in the original target quadrant was shortened, respectively, (38.93±8.33) s, (36.03±16.25) s, (29.85±9.27) s, and (29.84±10.16) s, respectively, and there was no significant difference among 4 groups (F=1.681, P=0.187). HE staining and Nissl staining showed that pathological changes such as edema, degeneration and necrosis were observed in the hippocampal CA1 area and CA2 area as well as dentate gyrus cells in rats exposed to arsenic in the acute phase.The higher the concentration of arsenic exposure, the more obvious the cell structure disorder was.However, 5 weeks after exposure, the pathological changes in hippocampal neurons in the arsenic-exposed group gradually returned to normal.Immunohistochemistry showed that the expressions of DCX in the CA1, CA2 and dentate gyrus of rats exposed to arsenic decreased significantly 24 h after arsenic exposure, especially in the 45 μg/L group.Five weeks after arsenic exposure, there was no expression in the hippocampal CA1-CA3 area, and there was still a small amount of expression in the dentate gyrus. Conclusions Postnatal low-concentration arsenic exposure may impair learning and abnormal germination of neurons in the hippocampal dentate gyrus may be the underlying mechanism. Key words: Arsenic exposure; Learning and memory; Hippocampus; Mechanism

Intranasal Administration of MSC‐Derived Exosomes Modulate Status Epilepticus Induced Abnormal Plasticity of Newly Born Neurons and Microglial Activation in the Hippocampus

Status epilepticus (SE) is a self‐sustaining seizure episode that causes adverse alterations in the hippocampus, which lead to a state of chronic epilepsy. We investigated the effects of mesenchymal stem cell (MSC)‐derived exosomes (small extracellular vesicles, EVs) to ameliorate the most prominent early changes induced by SE such as the abnormal plasticity of newly born neurons and activation of microglia. F344 rats were subjected to Kainic‐acid‐induced SE for 2 hours and then intranasally treated with a dose of exosomes (100 billion) or vehicle (VEH). In animals receiving VEH after SE (n=5), 20% of double cortin (DCX)‐positive newly born neurons displayed basal dendrites at 7 days post‐SE. Furthermore, the percentage of microglia with ramified morphology (resting microglia) was severely depleted in the CA1 subfield (6%) of these animals because of their activation. In contrast, in animals receiving EVs after SE (n=8), only 5% of DCX+ newly born neurons exhibited basal dendrites (p&lt;0.0001). Also, these animals displayed a higher percentage (20%) of ramified microglia than animals receiving VEH, implying a reduced level of activation (p&lt; 0.05). Thus, intranasal administration of MSC‐derived exosomes after SE is efficient for dampening the abnormal plasticity of newly born neurons and modulating microglial activation in the hippocampus. Basal dendrites promote abnormal connectivity between dentate granule cells and hilar/CA3 pyramidal neurons, as well as enhance granule cell to granule cell connectivity. Since both abnormal plasticity and neuroinflammation contribute to the occurrence of spontaneous seizures in the chronic phase, the results have significance.Support or Funding InformationSupported by a DOD grant to A.K.S.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Mechanism of mild hypothermia promoting nerve regeneration after traumatic brain injury in rats

Objective To investigate whether mild hypothermia can promote neurogenesis in the dentate gyrus of hippocampus and cognitive function recovery after traumatic brain injury (TBI) through inhibiting apoptosis of hippocampal neurons. Methods A total of 66 healthy adult Sprague-Dawley rats were randomly divided into sham group, TBI group and TBI+ hypothermia group, with 22 rats in each group. The rat TBI model was established using the fluid percussion device. The rats in TBI+ hypothermia group received 4-hour hypothermia therapy immediately after injury, with the target temperature of 33.5℃. Bromodeoxyuridine (BrdU) was injected into the rats' abdominal cavity to label the mitotic cells. The test of Morris water maze was used to evaluate the rats' spatial learning and memory capabilities. Immunofluorescence staining was used to observe the expression levels of BrdU, doublecortin (DCX), neuron specific nuclear protein (NeuN), cysteinyl aspartate specific proteinase 3 (caspase-3) and cleaved caspase-3 expressions in dentate gyrus of hippocampus at 7 days and 28 days after injury. Expressions apoptosis-related proteins including the factor associated suicide (FAS)/factor associated suicide ligand (FASL), B-cell lymphoma-2 (Bcl-2), caspase-3 and cleaved caspase-3 expressions were detected by Western blot assay. Results The water maze tests at 28 days after injury showed that compared with TBI group, the escape latency in TBI+ hypothermia group was significantly shorter [(24.2±5.9)s∶(18±4.1)s], and both the time in the target quadrant and the number of platform crossing were increased significantly [(24.9±6.5)s∶(31.7±5.2)s; (1.9±0.8) times∶(3.5±1.2)times](P<0.05). Compared with the sham group, in TBI group and TBI+ hypothermia group, the BrdU+ new-born cells in the dentate gyrus of hippocampus were significantly increased at 7 days after injury [(9.4±4.1)∶(33.4±3.8); (9.4±4.1)∶(45.8±5.6)], the BrdU+ /DCX+ new-born neurons were increased at 7 days after injury [(2.0±0.6)∶(9.6±1.6); (2.0±0.6)∶(19.2±3.7)], and the BrdU+ /NeuN+ mature neurons were increased at 28 days after injury [(2.6±1.0)∶(17.2±3.9); (2.6±1.0)∶(33.6±9.1)] (P<0.01). TBI group showed more obvious increase than the TBI+ hypothermia group (P<0.01). Moreover, compared with 7 days after injury, the number of BrdU+ cells at 28 days after injury was further increased in TBI+ hypothermia group but decreased in TBI group [(45.8±5.6)∶(58.8±9.2); (33.4±3.8)∶(22.0±3.5)](P<0.05 or <0.01). Compared with the sham group, the caspase-3+ NeuN+ and caspase-3+ NeuN+ apoptotic neurons were significantly increased at 7 days after injury in TBI group [(2.0±0.9)∶(11.6±2.6); (2.6±1.0)∶(10.2±2.9)] (P<0.05). Compared with the TBI group, the cleaved caspase-3+ NeuN+ apoptotic neurons were decreased in TBI+ hypothermia group [(6.6±2.0)∶(11.6±2.6)](P<0.05). Furthermore, compared with the TBI group, mild hypothermia might down-regulate the expression of FAS, FASL, cleaved caspase-3 and caspase-3 and up-regulate the expression of Bcl-2 in the hippocampus [(1.54±0.15) ∶(1.14±0.12); (1.06±0.04)∶(0.80±0.09); (0.84±0.03)∶(0.62±0.08); (0.93±0.06)∶(0.86±0.09); (0.71±0.01)∶(1.58±0.18)](P<0.05). Conclusions Mild hypothermia might inhibit apoptosis of hippocampal neurons through cleaved caspase-3, FAS/FASL and Bcl-2 pathways, thus improving the neurogenesis and maturation of neurons in the dentate gyrus of hippocampus and facilitating cognitive function recovery in rats. It indicates that the function of hypothermia in anti-apoptosis and neurogenesis and maturity of hippocampal neurons may have a potential role in predicting the prognosis of TBI patients. Key words: Hypothermia; Brain injuries; Hippocampus; Neuve regeneration; Apoptosis

Doublecortin X (DCX) serine 28 phosphorylation is a regulatory switch, modulating association of DCX with microtubules and actin filaments

Doublecortin X (DCX) plays essential roles in neuronal development via its regulation of cytoskeleton dynamics. This is mediated through direct interactions between its doublecortin (DC) domains (DC1 and DC2) with microtubules (MTs) and indirect association with actin filaments (F-ACT). While the regulatory role of the DCX C-terminus following DC2 (i.e. DCX residues 275–366) has been established, less is known of the possible contributions made by the DCX N-terminus preceding DC1 (i.e. DCX residues 1–44). Here, we assessed the influence of DCX Ser28 within the DCX N-terminus, on the association of DCX with MTs and F-ACT. We compared the cytoskeletal interactions of the DCX S28E phosphomimetic and DCX S28A phospho-resistant mutants and wild-type DCX. Immunoprecipitation and colocalisation analyses indicated increased association of DCX S28E with F-ACT but decreased interaction with MTs, and conversely enhanced DCX S28A association with MTs but decreased association with F-ACT. To evaluate the impact of DCX mutants on cytoskeletal filaments we performed fluorescence recovery after photobleaching (FRAP) studies on SiR-tubulin and β-actin-mCherry and observed comparable tubulin and actin exchange rates in the presence of DCX WT and DCX S28A. However, we observed faster tubulin exchange rates but slower actin exchange rates in the presence of DCX S28E. Moreover, DCX S28E enhanced the association with the actin-binding protein spinophilin (Spn) suggesting the shift to favour association with both F-ACT and Spn in the presence of DCX S28E. Taken together, our results highlight a new role for DCX S28 as a regulatory switch for cytoskeletal organisation.

Effects of sevoflurane on hippocampal neurogenesis in dentate gyrus of mice of different ages

Objective To evaluate the effects of sevoflurane on hippocampal neurogenesis in dentate gyrus (DG) of mice of different ages. Methods Ninety-six SPF healthy male C57BL/6 mice, aged 2 weeks, 6 weeks, 9 months and 20 months (24 mice for each age, 12 mice for each group), were divided into 2 groups (n=48 each) using a random number table method: control group (group C) and sevoflurane group (group S). Group S inhaled 3.0% sevoflurane for 2 h once a day for 3 consecutive days, while group C inhaled the mixture of air and O2.Six mice of each age were selected, and 5′-bromo-2′-deoxyuridine (BrdU) 50 mg/kg was intraperitoneally injected immediately before and after inhalation once a day for 3 consecutive days in two groups.Mice were sacrificed at 24 h after the last inhalation (T1), brains were removed and hippocampi isolated for determination of the number of nestin and doublecortin (DCX) positive cells in DG by immunohistochemistry.Mice were sacrificed at 4 weeks after the last inhalation (T2), brains were removed and hippocampi isolated for determination of the number of neuronal nuclei antigen (NeuN)/BrdU and glial fibrillary acid protein (GFAP)/BrdU positive cells by immunofluorescence. Results Compared with group C, the number of nestin and DCX positive cells was significantly reduced at T1, and the number of NeuN/BrdU and GFAP/BrdU positive cells was reduced at T2 in mice of 2 weeks and 20 months old (P 0.05). Conclusion Three percent sevoflurane can inhibit hippocampal neurogenesis in DG of immature and old mice and exerts no influence on hippocampal neurogenesis in DG of juvenile and adult mice. Key words: Anesthetic, inhalation; Age; Neurogenesis; Hippocampus

The macroscopic and microscopic effect of low-frequency whole body vibration after cerebral ischemia in rats

Whole body vibration (WBV) has been applied in stroke patients with uncertain effects on motor and sensory dysfunction, and its effects on neurogenesis have not been studied yet. In total, 144 rats were used for experiments with 29 rats excluded due to early attrition, exclusion by rCBF criterion or death of ischemia in acute phase. Rats included were divided into 3 groups: WBV group ( n = 50), control group ( n = 50) group, and sham group ( n = 15). To explore the effects of daily WBV on neurological behavior, brain structure, and neurogenesis after cerebral ischemia in rats for 4 weeks. Improvements in weight or comprehensive neurological deficits were not significantly different under WBV or control treatment, and the degrees of brain damage and the numbers of necrotic neurons in the ischemic cortex were similar in two groups. However, WBV markedly improved animals’ coordination from 14d to 28d ( P < 0.05) and muscle strength of the upper limbs at 21d and 28d ( P < 0.05 & P < 0.001) compared with the control group. WBV promoted the increase in the number of bromodeoxyuridine-positive (BrdU + ) cells at 3d ( P < 0.05) and 14d ( P < 0.001) and the number of BrdU+/nestin + cells at 14d ( P < 0.01) after ischemia when compared to the control group. The numbers of BrdU+/NeuN + cells at 21d and 28d ( P < 0.001) were enhanced by WBV treatment. WBV significantly promoted the proliferation of astrocytes and their neural processes thickening after 14d. The expression levels of neural markers, such as double cortin, microtubule-associated protein 2, and glial fibrillary acidic protein, were upregulated in the ipsilateral cortex at different time points. Low-frequency WBV showed inconspicuous improvements in behavioral performance and brain damage after cerebral ischemia, but showed the potential in improving coordination and muscle strength and promoted neurogenesis after long-term exposure.

Enhanced neurogenesis and possible synaptic reorganization in the piriform cortex of adult rat following kainic acid-induced status epilepticus.

Epileptic seizure has been reported to enhance adult neurogenesis and induce aberrant synaptic reorganization in the human dentate gyrus in the hippocampal formation. However, adult neurogenesis in the extrahippocampal regions has not been well studied. To investigate seizure-enhanced neurogenesis in the extrahippocampal regions, we performed histological and immunohistochemical as well as western blot analyses on the cerebrum of Sprague-Dawley rats (n = 51, male, 7 weeks old, body weight 250-300 g) treated with intraperitoneal injection of kainic acid (KA, 10 mg/kg) to induce status epilepticus (SE) (n = 36) or normal saline solution (n = 15) followed by 5'-bromo-2-deoxyuridine (BrdU) injection to label newborn cells. Even though severe neuronal damage was found in the piriform cortex of rats having SE, immunohistochemistry for double cortin (DCX) revealed an increase in the number of immature neurons in the piriform cortex. Double immunofluorescence staining demonstrated that DCX-positive cells in the piriform cortex were positive for both BrdU and neuronal nuclear antigen. Immunohistochemistry and western blotting revealed increased expressions of synaptophysin and postsynaptic density protein 95 in the piriform cortex of rat having SE. These results suggested the enhanced neurogenesis and possible synaptic reorganization in the piriform cortex of the KA-treated rat.

Dynamic microtubule association of Doublecortin X (DCX) is regulated by its C-terminus

Doublecortin X (DCX), known to be essential for neuronal migration and cortical layering in the developing brain, is a 40 kDa microtubule (MT)-associated protein. DCX directly interacts with MTs via its two structured doublecortin (DC) domains, but the dynamics of this association and the possible regulatory roles played by the flanking unstructured regions remain poorly defined. Here, we employ quantitative fluorescence recovery after photobleaching (FRAP) protocols in living cells to reveal that DCX shows remarkably rapid and complete exchange within the MT network but that the removal of the C-terminal region significantly slows this exchange. We further probed how MT organization or external stimuli could additionally modulate DCX exchange dynamics. MT depolymerisation (nocodazole treatment) or stabilization (taxol treatment) further enhanced DCX exchange rates, however the exchange rates for the C-terminal truncated DCX protein were resistant to the impact of taxol-induced stabilization. Furthermore, in response to a hyperosmotic stress stimulus, DCX exchange dynamics were slowed, and again the C-terminal truncated DCX protein was resistant to the stimulus. Thus, the DCX dynamically associates with MTs in living cells and its C-terminal region plays important roles in the MT-DCX association.

Maternal exposure to titanium dioxide nanoparticles during pregnancy and lactation alters offspring hippocampal mRNA BAX and Bcl-2 levels, induces apoptosis and decreases neurogenesis

IntroductionThe usage of Titanium dioxide nanoparticles (TiO2-NPs) covers a vast area in different fields ranging from cosmetics and food to the production of drugs. Maternal exposure to TiO2-NPs during developmental period has been associated with hippocampal injury and with a decrease in learning and memory status of the offspring. However, little is known about its injury mechanism. This paper describes the in vivo neurotoxic effects of TiO2-NPs on rat offspring hippocampus during developmental period. Material and methodsPregnant and lactating Wistar rats received intragastric TiO2-NPs (100mg/kg body weight) daily from gestational day (GD) 2 to (GD) 21 and postnatal day (PD) 2 to (PD) 21 respectively. Animals in the control groups received an equal volume of distilled water via gavage.At the end of the treatment process, offspring were deeply anesthetized and sacrificed. Then brains of each group were collected and sections of the rat offspring’s brains were stained using TUNEL staining (for detection of apoptotic cells) and immunostaining (for neurogenesis). Moreover, the right hippocampus (n=6 per each group) were removed from the right hemisphere for evaluating the expression of Bax and Bcl-2 level. ResultsResults of histopatological examination by TUNEL staining showed that maternal exposure to TiO2-NPs during pregnancy and lactation periods increased apoptotic cells significantly (P<0.01) in the offspring hippocampus. The immunolabeling of double cortin (DCX) protein as neurogenesis marker also showed that TiO2-NPs reduced neurogenesis in the hippocampus of the offspring (P<0.05). Moreover, in comparison with the control group, the mRNA levels of Bax and Bcl-2 in the TiO2-NPs group significantly increased and decreased, respectively (P<0.01). ConclusionThese findings provide strong evidence that maternal exposure to TiO2-NPs significantly impact hippocampal neurogenesis and apoptosis in the offspring. The potential impact of nanoparticle exposure for millions of pregnant mothers and their offspring across the world is potentially devastating.

The effect of maternal separation on the expression of glucocorticoid receptor and doublecortin in brain regions of young mice

Objective To observe the effect of maternal separation(MS)on the expression of glucocorticoid receptor(GR)and doublecortin(DCX)in hippocampus and prefrontal cortex of young mice. Methods Fifteen pregnant mice were randomly divided into 5 groups: the experimental group(3 d, 7 d, 14 d, 21 d)and the control group, n=3 in each group. Neonatal mice in experimental group were separated from mother for 3 h once a day, and the mice in control group were caged with mother without any processing after birth. All mice were weaned in postanal day 23. Separating female and male mice, and the male mice were killed and the brains were taken in postanal day 42. The expression and distribution of GR and DCX in hippocampus and prefrontal cortex were determined by immunohistochemical staining and Image J image analysis technique. Results The average optical density values of GR positive cells in hippocampus and prefrontal cortex, the average optical density values of DCX positive cells in hippocampal dentate gyrus subgranular zone in 3 d, 7 d, 14 d and 21 d group were(GR in hippocampus: 3 d((0.332±0.054)OD/μm2, 7 d(0.258±0.045)OD/μm2, 14 d(0.197±0.043)OD/μm2, 21 d(0.164±0.053)OD/μm2), (GR in prefrontal cortex: 3 d(0.356±0.043)OD/μm2, 7 d(0.278±0.041)OD/μm2, 14 d(0.220±0.039)OD/μm2, 21 d(0.157±0.034)OD/μm2)and(DCX in hippocampus: 3 d(0.193±0.020)OD/μm2, 7 d(0.146±0.033)OD/μm2, 14 d(0.110±0.032)OD/μm2, 21 d(0.060±0.017)OD/μm2)were lower than those in control group, which showed a decreasing trend with the increase of seperation time, and the average optical density values in 7 d, 14 d and 21 d group were obviously lower than those in control group(P<0.05). The average optical density values had statistical significance among the experimental groups(P <0.05). Conclusion MS can cause the decrease of GR in hippocampus and prefrontal cortex and reduce neurogenesis in the hippocampal dentate gyrus subgranular zone in young mice. It shows that the longer the seperation time, the greater the effect on hypothalamic-pituitary-adrenal(HPA) axis and neurogenesis. Key words: Maternal separation; Hippocampus; Prefrontal cortex; Glucocorticoid receptor; Doublecortin

Hippocampal expressions of metallothionein I/II and glycoprotein 96 in EAE-prone and EAE-resistant strains of rats.

Inflammatory demyelinating diseases such as multiple sclerosis and experimental autoimmune encephalomyelitis (EAE) are often followed by cognitive deficits associated with the neuronal injury, synaptic loss and altered neurogenesis within the hippocampus. Changes depend on the genetic and epigenetic factors that ensure the cellular and environmental homeostasis and regulate the interactions of immunocompetent, glial and neural cells. Owing to high impact of stress proteins on these processes, in this study we compared the protein content of interleukin-6, transforming growth factor-β1, metallothioneins I/II (MTs) and glycoprotein 96 (gp96) in the hippocampus of DA and AO rats that differ in the susceptibility to the induction of EAE, and tested the relationship of MTs and gp96 to granule neurons, glial cells and neural progenitors in different subfields of dentate gyrus. Rats were immunized with bovine brain homogenate emulsified in complete Freund's adjuvant or only with CFA. The data showed that acute attack of EAE in DA rats was followed by accumulation of IL-6, TGF-β1 and MTs proteins, by increased expression of MTs in molecular and granular cell layer, by reduced expression of gp96/granular cell, by apoptosis and by microgliosis with appearance of Iba-1+ cells, co-expressing MT I/II and gp96. Furthermore, in subgranular zone (SGZ) of DA rats an augmented number of GFAP+ precursors, but decreased number of doublecortin (DCX)+ neuroblasts and immature NeuN+ neurons were found, implying that in DA rats the neurogenesis was delayed or reduced. Besides, in SGZ of both strains several DCX+ and NeuN+ cells co-expressing gp96 and MT I/II were found.