Number Of BrdU-positive Cells Research Articles

Abstract 3091: Targeting low molecular weight (LMW) cyclin E-Cdk2 pathway for the prevention of breast cancer

Abstract Our laboratory was first to discover that full length cyclin E is proteolytically cleaved into low-molecular weight isoforms (LMW-E) specifically in tumor cells. The LMW-E isoforms are hyperactive and over-expressed in 70% of all triple negative breast cancer (TNBC) compared to only 30% of non-TNBC patients. Overexpression of LMW-E in transgenic mice induces mammary tumors similar to the basal subtype of breast cancer, which accounts for 70% of all human TNBCs. We have reported that Cdk2 deficient mice were completely resistant to LMW-cyclin E mediated mammary tumors. In this study, we have interrogated the ability of roscovitine in preventing the development of triple negative mammary tumors in the MMTV-LMW-E; p53+/- mouse model. Initially, a group of mice were treated from age 3 months with vehicle (DMSO/PBS, 40:60v/v, N = 8) or roscovitine (100 mg/kg, N = 8) once every 4 and 5 days alternatively by intraperitoneal injection and were followed up for mammary tumor development. Mice did not show any signs of toxicity. In this schedule of treatment, we did not find any difference in time to tumor formation or survival between the 2 groups (8.3 vs 7.9 months, P = 0.70, and 8.9 vs 8.4 months, P = 0.98, respectively). However, we found a 2.8-fold increase in tumor doubling time in the roscovitine-treated group indicating that roscovitine treatment decreased tumor growth (12.1+/-4.4 vs 4.4+/-0.9 days, P<0.05). This was due to a decreased proliferation as measured by BrdU immunohistochemistry (IHC) (13.6+/-0.9% vs 30.0 +/- 7.7%, P<0.05) and an increased apoptotic index as measured by cleaved caspase 3 IHC (49.6+/-9.4 CC3+ cells/x40 field vs 25.9 +/- 9.9, P<0.05). A second group of mice were treated at age 3 months with the same schedule but were killed after 2 months of treatment and the mammary glands were collected to investigate the effects of drug treatment on normal and premalignant mammary cells. The BrdU staining showed that 2 months of roscovitine treatment caused a 3-fold decrease in the number of BrdU positive cells (5.4+/-1.5% vs 14.6+/-3.8%, P<0.05) but did not completely block cyclin E-induced proliferation. This result may explain why the roscovitine-treated mice developed tumor with similar kinetics as the control group. Our ongoing work is to use gene expression profiling to determine the RNA changes induced by roscovitine for the discovery of potential biomarkers. The results from the first trial is suggestive that the mice need to be treated on a continued schedule to ensure that proliferation of the cyclin E overexpressing mammary tissue is completely inhibited. To this end, we are now treating the mice twice a day, 7 days on, 21 days off. We predict that the new treatment schedule will be more efficacious in inhibiting tumor formation. These preliminary data could support the development of a clinical trial testing cyclin-dependent kinase inhibitors for the prevention of human triple negative breast cancer especially those with LMW-E overexpression. Citation Format: Said Akli, Dong Yang, Tuyen N. Bui, Khandan Keyomarsi. Targeting low molecular weight (LMW) cyclin E-Cdk2 pathway for the prevention of breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3091. doi:10.1158/1538-7445.AM2015-3091

ACEA (a highly selective cannabinoid CB1 receptor agonist) stimulates hippocampal neurogenesis in mice treated with antiepileptic drugs

Hippocampal neurogenesis plays a very important role in learning and memory functions. In a search for best neurological drugs that protect neuronal cells and stimulate neurogenesis with no side effects, cannabinoids proved to be a strong group of substances having many beneficial properties. The aim of this study was to evaluate the impact of ACEA (arachidonyl-2′-chloroethylamide—a highly selective cannabinoid CB1 receptor agonist) combined with a classical antiepileptic drug sodium valproate (VPA) on neural precursor cells׳ proliferation and differentiation in the mouse brain. All experiments were performed on adolescent CB57/BL male mice injected i.p. with VPA (10mg/kg), ACEA (10mg/kg) and PMSF (30mg/kg) (phenylmethylsulfonyl fluoride—a substance protecting ACEA against degradation by the fatty-acid amidohydrolase) for 10 days. Next an acute response of proliferating neural precursor cells to ACEA and VPA administration was evaluated with Ki-67 staining (Time point 1). Next, in order to determine whether acute changes translated into long-term alterations in neurogenesis, proliferating cells were labeled with 5-bromo-2deoxyuridine (BrdU) followed by confocal microscopy used to determine the percentage of BrdU-labeled cells that showed mature cell phenotypes (Time point 2). Results indicate that ACEA with PMSF significantly increase the total number of Ki-67-positive cells when compared to the control group. Moreover, ACEA in combination with VPA increased the number of Ki-67-positive cells, whereas VPA administered alone had no impact on proliferating cells׳ population. Accordingly, neurogenesis study results indicate that the combination of ACEA+PMSF administered alone and in combination with VPA considerably increases the total number of BrdU-positive cells in comparison to the control group while ACEA+PMSF alone and in combination with VPA increased total numbers of BrdU-positive cells, newly born neurons and astrocytes as compared to VPA group but not to the control group. VPA administered alone decreased the number of newly born neurons with no significant impact on neurogenesis. These data provide substantial evidence that VPA administered chronically slightly decreases the proliferation and differentiation of newly born cells while combination of VPA+ACEA significantly increases the level of newborn neurons in the dentate subgranular zone.

Transiently impaired neurogenesis in MPTP mouse model of Parkinson's disease

It is still being debated whether neurogenesis in the subventricular zone (SVZ) is enhanced in response to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) injury in the adult mouse brain. Our previous studies provided evidence that MPTP induces apoptosis of migrating neuroblasts (neural progenitor cells, A cells) in the SVZ and rostral migratory stream (RMS). We investigated cellular kinetics in the adult SVZ and olfactory bulb (OB) after MPTP damage. Cells were labeled with bromodeoxyuridine (BrdU), and the effects of MPTP on the survival and fate of migrating and residing neuroblasts were evaluated. Two days after BrdU labeling and MPTP treatment, the number of BrdU-positive cells in the SVZ and OB of MPTP-treated mice was significantly lower than in the SVZ and OB of saline controls. Additionally, fewer BrdU-positive cells migrated to the OB of treated mice than to that of saline controls, and the cells that did migrate diffused radially into the granule cell layer (GCL) when observed at 7, 14, and 28 days. In the OB GCL, the differentiation of BrdU-positive cells into mature neurons significantly attenuated 14 and 28 days after MPTP injury. Moreover, the impaired neurogenesis was followed by a recovery of A cells in the SVZ and OB, suggesting activation of the self-repair process as a result of MPTP-induced depletion of BrdU-positive cells. Our findings clarify the kinetics underlying neurogenesis in MPTP-treated mice and may contribute to the development of an animal model of Parkinson's disease, and the demonstration of cellular kinetics in SVZ may also provide a new insight into assessing neurogenesis in MPTP-treated mouse.

Novel approach for the detection of tubular cell migration into the interstitium during renal fibrosis in rats.

BackgroundThe process of epithelial-mesenchymal transition (EMT), which is generally defined by phenotypic changes of injured tubules such as loss of epithelial markers or acquisition of mesenchymal markers, implies various activating steps, including proliferation, migration, and ability to produce extracellular matrix proteins. We established here a novel approach for the detection of tubular cell migration into the interstitium during renal fibrosis in vivo.ResultsUsing an osmotic pump, bromodeoxyuridine (BrdU) was continuously given to 7-week-old Wistar rats for 4 weeks, and BrdU-positive cells were detected by immunostaining. BrdU-positive cells were present in aquaporin-1-positive proximal tubules, but not in the interstitium of BrdU-treated rat kidneys. After unilateral ureteral obstruction (UUO), some BrdU-positive tubular cells protruded from the basement membrane and migrated into the interstitium. Interstitial BrdU-positive cells were co-localized with alpha-smooth muscle actin, fibroblast specific protein-1, vimentin, and type I collagen, but not with CD68 or CD3. No BrdU-positive cells were observed in the interstitium of sham-operated kidneys. The number of BrdU-positive cells migrating into the interstitium significantly increased and peaked at 8 days after UUO.ConclusionsLong-term BrdU labeling marked some of the proximal tubular cells and enabled us to detect tubular cell migration into the interstitium after UUO. This simple method might be useful to detect EMT in vivo.

Sigma‐1 receptor agonist and fluvoxamine rescue depressive behaviors in CaMKIV null mice

Sigma‐1 receptor (Sig‐1R) is a molecular chaperon regulating calcium transport from the neuronal endoplasmic reticulum to mitochondria (Shioda and Fukunaga, JBC 2012;287:23318). Recently Song et al (Int J Neuropsychopharmacol 2012;16:803) reported that CaMKIV null mice exhibit depressive behaviors and impaired neurogenesis as assessed by bromodeoxyuridine (BrdU) incorporation into newborn cells of the hippocampal dentate gyrus (DG). Moreover, the typical SSRI fluoxetine fails to induced DG neurogenesis and lacks of its anti‐depressive effects in CaMKIV null mice. Here, we addressed whether Sig‐1R agonist (SA4503) and SSRI (fluvoxamine) with affinity for Sig‐1R rescue the depressive behaviors in CaMKIV null mice. Treatment with SA4503 or fluvoxamine for 14 days completely rescued the depressive behaviors in CaMKIV null mice. By contrast, treatment with paroxetine, which lacks affinity for Sig‐1R, did not alter these behaviors. Reduced numbers of BrdU‐positive cells and decreased BDNF mRNA expression seen in the DG of CaMKIV null mice were significantly restored by chronic Sig‐1R stimulation with SA4503 or fluvoxamine. We previously reported that Sig‐1R stimulation with SA4503 and fluvoxamine promotes ATP production through increased mitochondrial calcium transport (Tagashira et al., BBA2013;1830:3082). As expected, the reduced ATP production in the DG of CaMKIV null mice was improved by chronic Sig‐1R stimulation. Taken together, Sig‐1R stimulation rescues the SSRI‐resistant depressive behaviors observed in CaMKIV null mice, suggesting attractive therapeutics for SSRI‐resistant depressive patients.

The effects of acute and chronic administration of phosphatidylserine on cell proliferation and survival in the dentate gyrus of adult and middle-aged rats

Phosphatidylserine (PS) is an acidic phospholipid that is widely used as an alternative and/or complementary treatment of cognitive impairments. We hypothesize that these changes may be attributable, at least in part, to alterations in hippocampal neurogenesis. The aim of the present study was to investigate the effects of acute and chronic PS administration on hippocampal cell proliferation and survival in adult (5 months old) and middle-aged (12 months old) male Wistar rats. PS was injected daily (50mg/kg, i.p.) during 7 days (acute experiment) or 21 days (chronic experiment). To label newly generated cells, rats received a single BrdU injection (200mg/kg, i.p.) one day before PS treatment. The object recognition test was performed, and the rats were perfused. The brains were removed and processed with immunohistochemistry techniques for Ki-67 (cell proliferation) and BrdU (cell survival). The acute and chronic regimens were unable to promote cognitive improvement in either age group in the object recognition test. The analysis of cell proliferation showed a significant increase in the number of Ki-67-positive cells after acute and chronic PS administration in both age groups. The analysis of cell survival showed that acute and chronic PS administration increased the number of BrdU-positive cells only in adult animals.

MMP-9 inhibition facilitates amacrine cell loss after ouabain-induced retinal damage

Retinal ischemia is a common risk factor for visual impairment and blindness. Two common changes after retinal ischemia are retinal ganglion cell (RGC) loss and Müller glial cell (MGC)-mediated endogenous repair. Matrix metalloproteinase 9 (MMP-9) has been shown to be responsible to RGC death. However, the effects of MMP-9 on the loss of other neurons and the reactivity of MGCs after retinal injury remain unclear. Ouabain, a Na/K-ATPase inhibitor, was injected into the vitreous body of rat eyes to induce cell death in the inner nuclear layer (INL). MMP-9 expression and activation in the retinas were examined by gelatin zymography and immunohistochemistry. The role of MMP-9 inhibitor (MMP-9i) in ouabain-treated retinas was assessed. After ouabain injection, there was an upregulation of MMP-9 activity in the inner retinas, and the activation of MMP-9 reached a maximum at 2 day. Unexpectedly, MMP-9i enhanced the thinning of the INL, the loss of Calbindin D-28k-positive cells and Syntaxin-positive amacrine cells (ACs) in the INL and decreased levels of Calbindin D-28k protein, while leaving the outer nuclear layer (ONL) unchanged. In addition, MMP-9i led to a minor increase in the number of BrdU positive cells that did not express GS in the INL. Collectively, these results revealed that the inhibition of MMP-9 activity facilitated AC loss and promoted the generation of MGC-derived cells in ouabain-treated retinas, which indicates that treating retinal diseases with drugs that inhibit MMP-9 activity should be considered with caution.

Increasing Proliferation of Intrinsic Tubular Cells after Renal Ischemia-reperfusion Injury in Adult Rat.

The kidney is capable of regeneration following injury. However, whether renal stem/progenitor cells contribute to the repair process after injury, as well as the origin of the cells that repair and replace damaged renal tubule cells remains debated. Therefore, better understanding of the repair process will be critical to developing new strategies for the treatment of acute renal failure. Using an ischemia-reperfusion injury mode and an immunocytochemistry method, we counted the number of BrdU-positive cells in damged regions at different durations of reperfusion. We found that BrdU, a cell proliferative marker, was mainly incorporated in the tubular cells of both medulla and cortex 1 day after reperfusion. The number of BrdU-positive cells reached a peak at 3 days and lasted for two months after injury. BrdU-positive cells were barely found in the renal glomerulus and the parietal layer of Bowman's capsule after injury, and only a few were found in the intersititium. PAX2, an embryonic renal marker, was also increased in renal tubule cells. Confocal images show that BrdU-positive cells co-expressed PAX2, but not the activated form of caspase-3, a cell death marker. Our data suggest that renal stem-like cells or dedifferentiation of surviving renal tubular cells in both the medulla and cortex may predominantly contribute to the repair process after renal ischemia-reperfusion injury in rat.

Prediabetes and type 2 diabetes implication in central proliferation and neurogenesis

Type 2 diabetes (T2D) is an important risk factor for developing dementias, including Alzheimer's disease (AD). Hyperinsulinemia and glucose intolerance, as features of T2D, might increase the neurodegeneration process, synaptic loss and brain atrophy, leading to cognitive impairment observed in AD. Also, adult neurogenesis seems to be impaired in AD models. Therefore, we have studied morphological alterations, cell proliferation and neurogenesis in the central nervous system (CNS) from a classical model of T2D, the db/db mouse, and in a prediabetes insulin-resistant model, obtained after long-term high fat diet (HFD) administration to C57Bl/6 mice. Db/db mice showed an age-dependent cortical and hippocampal atrophy, whereas in HFD mice cortex and hippocampus were preserved. Neurogenesis and cell proliferation were increased in young db/db mice, when compared with control mice, whereas no differences were detected in the prediabetic model. We also detected significant correlations between metabolic parameters and central atrophy, altered proliferation and neurogenesis in the central nervous system. Altogether these data support that glycaemia control in elderly patients, could help to control central alterations and improve dementia prognosis.

Stimulation of Sigma-1 Receptor Ameliorates Depressive-like Behaviors in CaMKIV Null Mice.

Sigma-1 receptor (Sig-1R) is a molecular chaperone regulating calcium efflux from the neuronal endoplasmic reticulum to the mitochondria. Calcium/calmodulin-dependent protein kinase IV (CaMKIV) null mice exhibit depressive-like behaviors and impaired neurogenesis as assessed by bromodeoxyuridine (BrdU) incorporation into newborn cells of the hippocampal dentate gyrus (DG). Here, we demonstrate that chronic stimulation of Sig-1R by treatment with the agonist SA4503 or the SSRI fluvoxamine for 14days improves depressive-like behaviors in CaMKIV null mice. By contrast, treatment with paroxetine, which lacks affinity for Sig-1R, did not alter these behaviors. Reduced numbers of BrdU-positive cells and decreased brain-derived neurotrophic factor (BDNF) mRNA expression and protein kinase B (Akt; Ser-473) phosphorylation seen in the DG of CaMKIV null mice were significantly rescued by chronic Sig-1R stimulation. Interestingly, reduced ATP production observed in the DG of CaMKIV null mice was improved by chronic Sig-1R stimulation. Such stimulation also improved hippocampal long-term potentiation (LTP) induction and maintenance, which are impaired in the DG of CaMKIV null mice. LTP rescue was closely associated with both increases in calcium/calmodulin-dependent protein kinase II (CaMKII) autophosphorylation and GluA1 (Ser-831) phosphorylation. Taken together, Sig-1R stimulation by SA4503 or fluvoxamine treatment increased hippocampal neurogenesis, which is closely associated with amelioration of depressive-like behaviors in CaMKIV null mice.

Effects of donepezil, an acetylcholinesterase inhibitor, on neurogenesis in a rat model of vascular dementia

Vascular dementia (VaD) is the second most common form of dementia caused by cerebrovascular disease. Several recent reports demonstrated that cholinergic deficits are implicated in the pathogenesis of VaD and that cholinergic therapies have shown improvement of cognitive function in patients with VaD. However, the precise mechanisms by which donepezil achieves its effects on VaD are not fully understood. Donepezil hydrochloride is an acetylcholinesterase inhibitor (AChEI) currently used for the symptomatic treatment of Alzheimer's disease (AD). Several lines of evidence have demonstrated that AChEIs such as donepezil promote neurogenesis in the central nervous system. We investigated whether donepezil regulated hippocampal neurogenesis after bilateral common carotid artery occlusion (BCCAO) in rats, a commonly used animal model of VaD.To evaluate the effect of donepezil on neurogenesis, we orally treated rats with donepezil (10mg/kg) once a day for 3weeks, and injected BrdU over the same 3-week period to label newborn cells. The doses of donepezil that we used have been reported to activate cholinergic activity in rats. After 3weeks, a water maze task was performed on these rats to test spatial learning, and a subsequent histopathological evaluation was conducted. Donepezil improved memory impairment and increased the number of BrdU-positive cells in the dentate gyrus (DG) of BCCAO animals. These results indicated that donepezil improves cognitive function and enhances the survival of newborn neurons in the DG in our animal model of VaD, possibly by enhancing the expression of choline acetyltransferase and brain-derived neurotropic factor.

Slit2 promotes tumor growth and invasion in chemically induced skin carcinogenesis

Slit, a neuronal guidance cue, binds to Roundabout (Robo) receptors to modulate neuronal, leukocytic, and endothelial migration. Slit has been reported to have an important effect on tumor growth and metastasis. In the current study, we evaluated the role of Slit2 in skin tumor growth and invasion in mice using a two-step chemical carcinogenesis protocol. We found that Slit2 expression correlated with the loss of basement membrane in the samples of human skin squamous cell carcinoma at different stages of disease progression. Slit2-Tg mice developed significantly more skin tumors than wild-type mice. Furthermore, the skin tumors that occurred in Slit2-Tg mice were significantly larger than those in the wild-type mice 10 weeks after 7,12-dimethylbenz[a]anthracene initiation until the end of the experiment. We also found that pathological development of the wild-type mice was delayed compared with that of Slit2-Tg mice. To further investigate the mechanism of increasing tumors in Slit2-Tg mice, we analyzed the expression of 5-bromo-2′-deoxyuridine (BrdU) in mouse skin lesions and found that the number of BrdU-positive cells and microvessel density in skin lesions were significantly higher in Slit2-Tg mice than in wild-type mice. Histological staining of PAS and type IV collagen and the colocalization of Slit2 and type IV collagen demonstrated varying degrees of loss of the basement membrane in the skin lesions from Slit2-Tg mice that were at the stage of carcinoma in situ. However, the basement membrane was well defined in the wild-type mice. In addition, MMP2, but not MMP9, was upregulated in the skin tissue of Slit2-Tg mice. Interruption of Slit2–Robo1 signaling by the antibody R5 significantly repressed the invasive capability of the squamous cell carcinoma cell line A431. Taken together, our findings reveal that Slit2 promotes DMBA/TPA-induced skin tumorigenesis by increasing cell proliferation, microvessel density, and invasive behavior of cutaneous squamous cell carcinoma, along with loss of basement membrane, by upregulation of MMP2 expression.

Laminin α1 Regulates Age-Related Mesangial Cell Proliferation and Mesangial Matrix Accumulation through the TGF-β Pathway

Laminin α1 (LAMA1), a subunit of the laminin-111 basement membrane component, has been implicated in various biological functions in vivo and in vitro. Although LAMA1 is present in kidney, its roles in the kidney are unknown because of early embryonic lethality. Herein, we used a viable conditional knockout mouse model with a deletion of Lama1 in the epiblast lineage (Lama1(CKO)) to study the role of LAMA1 in kidney development and function. Adult Lama1(CKO) mice developed focal glomerulosclerosis and proteinuria with age. In addition, mesangial cell proliferation was increased, and the mesangial matrix, which normally contains laminin-111, was greatly expanded. In vitro, mesangial cells from Lama1(CKO) mice exhibited significantly increased proliferation compared with those from controls. This increased proliferation was inhibited by the addition of exogenous LAMA1-containing laminin-111, but not by laminin-211 or laminin-511, suggesting a specific role for LAMA1 in regulating mesangial cell behavior. Moreover, the absence of LAMA1 increased transforming growth factor (TGF)-β1-induced Smad2 phosphorylation, and inhibitors of TGF-β1 receptor I kinase blocked Smad2 phosphorylation in both control and Lama1(CKO) mesangial cells, indicating that the increased Smad2 phosphorylation occurred in the absence of LAMA1 via the TGF-β1 receptor. These findings suggest that LAMA1 plays a critical role in kidney function and kidney aging by regulating the mesangial cell population and mesangial matrix deposition through TGF-β/Smad signaling.

Extended Effect of Chronic Social Defeat Stress in Childhood on Behaviors in Adulthood

Individuals exposed to social stress in childhood are more predisposed to developing psychoemotional disorders in adulthood. Here we use an animal model to determine the influence of hostile social environment in adolescence on behavior during adult life. One-month-old adolescent male mice were placed for 2 weeks in a common cage with an adult aggressive male. Animals were separated by a transparent perforated partition, but the adolescent male was exposed daily to short attacks from the adult male. After exposure to social stress, some of the adolescent mice were placed for 3 weeks in comfortable conditions. Following this rest period, stressed young males and adult males were studied in a range of behavioral tests to evaluate the levels of anxiety, depressiveness, and communicativeness with an unfamiliar partner. In addition, adult mice exposed to social stress in adolescence were engaged in agonistic interactions. We found that 2 weeks of social stress result in a decrease of communicativeness in the home cage and diminished social interactions on the novel territory. Stressed adolescents demonstrated a high level of anxiety in the elevated plus-maze test and helplessness in the Porsolt test. Furthermore, the number of dividing (BrdU-positive) cells in the subgranular zone of the dentate gyrus was significantly lower in stressed adolescents. After 3 weeks of rest, most behavioral characteristics in different tests, as well as the number of BrdU-positive cells in the hippocampus, did not differ from those of the respective control mice. However, the level of anxiety remained high in adult males exposed to chronic social stress in childhood. Furthermore, these males were more aggressive in the agonistic interactions. Thus, hostile social environment in adolescence disturbs psychoemotional state and social behaviors of animals in adult life.

Valproic acid decreases cell proliferation and migration in the cerebellum of zebrafish larvae

Valproic acid (VPA) is used as an antiepileptic drug or mood stabilizer. Recent studies have shown that exposure to VPA during embryonic development alters neural progenitor cell proliferation. The main goal of this investigation was to elucidate the effects of VPA on cell proliferation in the cerebellum of zebrafish larvae using immunohistochemistry. 5-bromo-2'-deoxyuridine (BrdU) and proliferating cell nuclear antigen (PCNA)-labeled cells were mostly found in the ventral cerebellum proliferation zone and external granular layer of the cerebellum from 5 days postfertilization zebrafish immediately after treatment with BrdU for 12 h. Subsequently, BrdU-labeled cells were mostly found in the medial zone of the cerebellar plate 48 h after terminating BrdU treatment. Pretreatment with 2-mM VPA for 3 h prior to BrdU exposure reduced the number of BrdU-labeled cells both immediately and 48 h after terminating BrdU exposure. Posttreatment with VPA following BrdU exposure reduced the migration of BrdU-labeled cells, represented by a decreased number of BrdU-positive cells in the medial zone of the cerebellar plate. These results suggest that VPA may delay brain development by hindering cell proliferation and migration during the early developmental period.

Beneficial Effects of Melatonin Combined with Exercise on Endogenous Neural Stem/Progenitor Cells Proliferation after Spinal Cord Injury

Endogenous neural stem/progenitor cells (eNSPCs) proliferate and differentiate into neurons and glial cells after spinal cord injury (SCI). We have previously shown that melatonin (MT) plus exercise (Ex) had a synergistic effect on functional recovery after SCI. Thus, we hypothesized that combined therapy including melatonin and exercise might exert a beneficial effect on eNSPCs after SCI. Melatonin was administered twice a day and exercise was performed on a treadmill for 15 min, six days per week for 3 weeks after SCI. Immunohistochemistry and RT-PCR analysis were used to determine cell population for late response, in conjunction with histological examination and motor function test. There was marked improvement in hindlimb function in SCI+MT+Ex group at day 14 and 21 after injury, as documented by the reduced size of the spinal lesion and a higher density of dendritic spines and axons; such functional improvements were associated with increased numbers of BrdU-positive cells. Furthermore, MAP2 was increased in the injured thoracic segment, while GFAP was increased in the cervical segment, along with elevated numbers of BrdU-positive nestin-expressing eNSPCs in the SCI+MT+Ex group. The dendritic spine density was augmented markedly in SCI+MT and SCI+MT+Ex groups. These results suggest a synergistic effect of SCI+MT+Ex might create a microenvironment to facilitate proliferation of eNSPCs to effectively replace injured cells and to improve regeneration in SCI.

Effect of the N‐methyl‐D‐aspartate NR2B subunit antagonist ifenprodil on precursor cell proliferation in the hippocampus

The N-methyl-D-aspartate (NMDA) receptor, one of the ionotropic glutamate receptor, plays important physiological and pathological roles in learning and memory, neuronal development, acute and chronic neurological diseases, and neurogenesis. This work examines the contribution of the NR2B NMDA receptor subunit to adult neurogenesis/cell proliferation under physiological conditions and following an excitotoxic insult. We have previously shown in vitro that a discrete NMDA-induced, excitotoxic injury to the hippocampus results in an increase in neurogenesis within the dentate gyrus. Here we have characterized adult neurogenesis or proliferation, using BrdU, in an in vivo model of excitotoxic injury to the CA1 subfield of the hippocampus. We demonstrate a peak in neural stem cell proliferation/neurogenesis between 6 and 9 days after the excitotoxic insult. Treatment with ifenprodil, an NR2B subunit-specific NMDA receptor antagonist, without prior injury induction, also increased the number of BrdU-positive cells within the DG and posterior periventricle, indicating that ifenprodil itself could modulate the rate of proliferation. Interestingly, though, the increased level of cell proliferation did not change significantly when ifenprodil was administered following an excitotoxic insult. In conclusion, our results suggest and add to the growing evidence that NR2B subunit-containing NMDA receptors play a role in neural stem cell proliferation.

Interleukin 18 accelerates the hepatic cell proliferation in rat liver regeneration after partial hepatectomy

Interleukin 18 (IL-18) is a proinflammatory cytokine with an ability to accelerate cell proliferation through activating other factors. However, little is yet understood of the role of IL-18 in the regulation of liver regeneration (LR). To study the effect of IL-18 on LR, the gene expression profiles of hepatocytes isolated from rat regenerative liver were determined by Rat Genome 230 2.0 microarray. Next, the synergistic effects of genes associated to IL-18 pathway were analyzed by expression profile function Et. Then, the expression level of IL-18 was examined by RT-PCR and ELISA. Finally, the effect of IL-18 on hepatocyte proliferation was detected by injecting recombinant rat IL-18 (rrIL-18) into rats immediately after partial hepatectomy (PH) and the rate of hepatocyte proliferation was detected by BrdU labeling. The microarray result showed that the expressions of 13 genes of IL-18 pathway and 49 cell proliferation genes regulated by the pathway were significantly altered at transcriptional level. The Et values of three branches of IL-18 pathway, NF-κB, p38 and JNK, were markedly enhanced during the priming and progressing phases of rat LR. The mRNA level of IL-18 was significantly elevated at 2 and 36h, and its level in plasma was also significantly increased at 2h, and reached the peaks at 12h and 48h after PH (p<0.05). The number of BrdU positive cells was dramatically increased in rats treated with IL-18 compared to PBS control group (p<0.01). In conclusion, IL-18 promotes rat hepatocyte proliferation in the LR priming and progressing phases after PH.

Effects of High Salt-Exposure on the Development of Retina and Lens in 5.5-Day Chick Embryo

Background/Aims: Excess maternal salt intake during pregnancy may alter fetal development. However, our knowledge on how an increased salt intake during pregnancy influences fetal eye development is limited. In this study, we investigated the effects of high-salt treatment on the developing eyes in chick embryos, especially focusing on the development of the retina and the lens. Methods: 5.5-day chick embryos were exposed to 280mosm/l (n=17), or 300mosm/l (n=16) NaCl. The treated embryos were then incubated for 96 hours before they were fixed with 4% paraformaldehyde for H&E staining, whole-mount embryo immunostaining and TUNEL staining. BrdU and PH3 incorporation experiments were performed on the chick embryos after high-salt treatment. RT-PCR analyses were conducted from chick retina tissues. Results: We demonstrated that high-salt treatment altered the size of eyes in chick embryos, induced malformation of the eyes and impaired the development of the lens and the retina. We found an impaired expression of Paired box 6 (PAX6) and neuronal cells in the developing retina as revealed by neurofilament immunofluorescent staining. There was a reduction in the number of BrdU-positive cells and PH3-positive cells in the retina, indicating an impaired cell proliferation with high-salt treatment. High-salt treatment also resulted in an increased number of TUNEL-positive cells in the retina, indicating a higher amount of cell death. RT-PCR data displayed that the expression of the pro-apoptotic molecule nerve growth factor (NGF) in chick retina was increased and CyclinD1 was reduced with high-salt treatment. The size of the lens was reduced and Pax6 expression in the lens was significantly inhibited. High salt-treatment was detrimental to the migration of neural crest cells. Conclusion: Taken together, our study demonstrated that high-salt exposure of 5.5-day chick embryos led to an impairment of retina and lens development, possibly through interfering with Pax6 expression.

Proliferation, differentiation and amyloid-β production in neural progenitor cells isolated from TgCRND8 mice

The amyloid precursor protein (APP) and amyloid-β (Aβ) peptide play central roles in the pathology and etiology of Alzheimer’s disease. Amyloid-induced impairments in neurogenesis have been investigated in several transgenic mouse models but the mechanism of action remains to be conclusively demonstrated. The changes in neurogenesis during this transition of increasing Aβ levels and plaque formation were investigated in the present study. We found that the proliferation of newborn cell in the dentate gyrus was enhanced prior to elevations in soluble Aβ production as well as amyloid deposition in 5-week-old TgCRND8 mice, which are well-established Alzheimer’s disease models, compared to non-transgenic (Non-Tg) mice. The number of BrdU-positive cells remained higher in TgCRND8 vs Non-Tg mice for a period of 8weeks. The numbers of BrdU/NeuN-positive cells were not significantly different in TgCRND8 compared to Non-Tg mice. A significant decrease in BrdU/GFAP but not in BrdU/S100β was found in Tg vs Non-Tg at 6-weeks of age. In addition, a unique observation was made using isolated neuroprogenitor cells from TgCRND8 mice which were found to be less viable in culture and produced substantial amounts of secreted Aβ peptides. This suggests that the proliferation of neural progenitors in vivo may be modulated by high levels of APP expression and the resulting Aβ generated directly by the progenitor cells. These findings indicate that cell proliferation is increased prior to Aβ deposition and that cell viability is decreased in TgCRND8 mice over time.