Experimental 'jet lag' inhibits adult neurogenesis and produces long-term cognitive deficits in female hamsters

Background

Circadian disruptions through frequent transmeridian travel, rotating shift work, and poor sleep hygiene are associated with an array of physical and mental health maladies, including marked deficits in human cognitive function. Despite anecdotal and correlational reports suggesting a negative impact of circadian disruptions on brain function, this possibility has not been experimentally examined.

Methodology/Principal Findings

In the present study, we investigated whether experimental ‘jet lag’ (i.e., phase advances of the light∶dark cycle) negatively impacts learning and memory and whether any deficits observed are associated with reductions in hippocampal cell proliferation and neurogenesis. Because insults to circadian timing alter circulating glucocorticoid and sex steroid concentrations, both of which influence neurogenesis and learning/memory, we assessed the contribution of these endocrine factors to any observed alterations. Circadian disruption resulted in pronounced deficits in learning and memory paralleled by marked reductions in hippocampal cell proliferation and neurogenesis. Significantly, deficits in hippocampal-dependent learning and memory were not only seen during the period of the circadian disruption, but also persisted well after the cessation of jet lag, suggesting long-lasting negative consequences on brain function.

Conclusions/Significance

Together, these findings support the view that circadian disruptions suppress hippocampal neurogenesis via a glucocorticoid-independent mechanism, imposing pronounced and persistent impairments on learning and memory.     

Repairing brain after stroke: a review on post-ischemic neurogenesis

Stroke is devastating as currently no therapies are available that can prevent stroke-induced neurological dysfunction in humans. With the recent observations that acute insults to adult brain stimulate new neuronal formation in various species of animals, optimism is building for a possible regeneration of stroke-damaged brain. This article reviewed the advances in the understanding of the molecular mechanisms of the various steps of neurogenesis with an emphasis on the endogenous mediators and exogenous promoters of neural progenitor proliferation, migration and survival in the post-ischemic adult brain.     

Sox11 promotes endogenous neurogenesis and locomotor recovery in mice spinal cord injury

We introduced a lentiviral vector containing the Sox11 gene into injured spinal cords of mice to evaluate the therapeutic potential of Sox11 in spinal cord injury. Sox11 markedly improved locomotor recovery after spinal cord injury and this recovery was accompanied by an up-regulation of Nestin/Doublecortin expression in the injured spinal cord. Sox11 was mainly located in endogenous neural stem cells lining the central canal and in newly-generated neurons in the spinal cord. In addition, Sox 11 significantly induced expressions of BDNF in the spinal cords of LV-Sox11-treated mice. We concluded that Sox11 induced activation of endogenous neural stem cells into neuronal determination and migration within the injured spinal cord. The resultant increase of BDNF at the injured site might form a distinct neurogenic niche which induces a final neuronal differentiation of these neural stem cells. Enhancing Sox11 expression to induce neurogenic differentiation of endogenous neural stem cells after injury may be a promising strategy in restorative therapy after SCI in mammals.     

Cortical propagation tracks functional recovery after stroke

Stroke is a debilitating condition affecting millions of people worldwide. The development of improved rehabilitation therapies rests on finding biomarkers suitable for tracking functional damage and recovery. To achieve this goal, we perform a spatiotemporal analysis of cortical activity obtained by wide-field calcium images in mice before and after stroke. We compare spontaneous recovery with three different post-stroke rehabilitation paradigms, motor training alone, pharmacological contralesional inactivation and both combined. We identify three novel indicators that are able to track how movement-evoked global activation patterns are impaired by stroke and evolve during rehabilitation: the duration, the smoothness, and the angle of individual propagation events. Results show that, compared to pre-stroke conditions, propagation of cortical activity in the subacute phase right after stroke is slowed down and more irregular. When comparing rehabilitation paradigms, we find that mice treated with both motor training and pharmacological intervention, the only group associated with generalized recovery, manifest new propagation patterns, that are even faster and smoother than before the stroke. In conclusion, our new spatiotemporal propagation indicators could represent promising biomarkers that are able to uncover neural correlates not only of motor deficits caused by stroke but also of functional recovery during rehabilitation. In turn, these insights could pave the way towards more targeted post-stroke therapies.     

Conditional RNAi in mice

RNA interference (RNAi)-mediated gene knockdown has developed into a routine method to assess gene function in cultured mammalian cells in a fast and easy manner. For the use of RNAi in mice, short hairpin (sh) RNAs expressed stably from the genome are a fast alternative to conventional knockout approaches. We developed a strategy for complete or conditional gene knockdown in mice, where the Cre/loxP system is used to activate RNAi in a time and tissue dependent manner. Alternatively doxycycline controlled shRNA expression vectors can be used for conditional gene silencing. Single copy RNAi constructs are placed into the Rosa26 locus of ES cells by recombinase mediated cassette exchange and transmitted through the germline of chimeric mice. The shRNA transgenic offspring can be either directly used for phenotypic analysis or are further crossed to a Cre transgenic strain to activate conditional shRNA vectors. The site specific insertion of single copy shRNA vectors allows the expedite and reproducible production of knockdown mice and provides an easy and fast approach to assess gene function in vivo.     

NK cell depletion and recovery in SCID mice treated with anti-NK1.1 antibody

The anti-NK1.1 antibody produced by PK136 hybridoma cell line administered subcutaneously to SCID mice effectively decreased the level of peripheral blood NK cells and weight of the spleen for 3-4 days. The antibody treatment did not harm the general state of the animal, and may be practically applied in xenograft experiments.     

Conditional genome alteration in mice

The recent ability to inactivate specific genes in mice has significantly accelerated our understanding of molecular, cellular, and even behavioral aspects of normal and disease processes. However, this ability has also demonstrated the extreme complexity of genetic determination in mammals, in particular, that genes in the same family or pathway can be functionally redundant and that a given gene often has multiple roles. Thus, inactivation of a gene often does not indicate its complete spectrum of functions. To circumvent this problem, many new tools and novel applications of classic techniques have been developed to place spatial and temporal restrictions on the genomic alterations. These approaches include chimera and mosaic studies, organ transplantation, complementation assays, dominant negative mutants, conditional gene knockouts, and lineage-specific gene rescue. Not only has this opened up more sophisticated ways to make genomic alterations, but it has provided the opportunity to create animal models for sporadic human genetic diseases. BioEssays 20 :200–208, 1998. © 1998 John Wiley & Sons, Inc.     

Progesterone and allopregnanolone improves stroke outcome in male mice via distinct mechanisms but neither promotes neurogenesis

Based on the outcome of a number of experimental studies, progesterone (PROG) holds promise as a new therapy for stroke. To understand more about the mechanisms involved, we administered PROG (or the major metabolite, allopregnanolone, ALLO), intra‐peritoneally, for a period of 24 h after transient middle cerebral artery occlusion to male mice variably expressing intracellular progesterone receptors (iPR) A/B. Effects on infarct volume and neurogenesis were then assessed up to 1 month later. Predictably, infarct volume in wild‐type mice receiving either drug was significantly smaller. However, mice heterozygous for iPRs A/B showed protection by ALLO but not by PROG. There was robust amplification of cell division in the wall of the lateral ventricle on the injured side of the brain, these cells migrated into the striatum and lateral cortex, and a significant number survived for at least 3 weeks. However, very few doublecortin‐positive cells emerged from the subventricular zone and subsequent expression of NeuN in these newborn neurons was extremely rare. Neither PROG nor ALLO amplified the rate of neurogenesis, suggesting that the long‐term benefits of acute drug administration results from tissue preservation.

     

Homogeneous repair of nuclear genes after experimental stroke

The repair of oxidative DNA lesions (ODLs) in the nucleus of ischemic cortical brain cells was examined following experimentally induced stroke by occluding the right middle cerebral artery and both common carotid arteries for 60-90 min followed by reperfusion in male long-Evans hooded rats. The control group consisted of sham-operated animals undergoing the same surgery without vessel occlusion. Using a gene-specific assay based upon the presence of Escherichia coli Fpg protein-sensitive sites, we noted that animals with stroke exhibited six and four ODLs per gene in the actin and DNA polymerase-beta genes, respectively. This was increased from one per four copies of each gene in the sham-operated control (p < 0.01). One half of the initial ODLs was repaired within 30 min, and 83% of them were repaired as early as 45 min of reperfusion. There was no further increase when gene repair was measured again at 2 h of reperfusion. The rates of active repair within 45 min of reperfusion were the same in these two genes (p = 0.103, ANOVA). BrdU (10 mg/kg) was administered via intraperitoneal injection at least one day before surgery. We observed that there was no significant incorporation of BrdU triphosphates into genomic DNA during active repair, but there were significant amounts of BrdU triphosphate in nuclear DNA after active repair. The result indicates that genomic repair of ODLs in the brain did not significantly incorporate BrdU, and the initiation of neurogenesis probably starts after the completion of repair in the brain.     

Conditional induction of ovulation in mice

Follicle-stimulating hormone controls the maturation of mammalian ovarian follicles. In excess, it can increase ovulation (egg production). Reported here is a transgenic doxycycline-activated switch, tested in mice, that produced more FSHB subunit (therefore more FSH) and increased ovulation by the simple feeding of doxycycline (Dox). The transgenic switch was expressed selectively in pituitary gonadotropes and was designed to enhance normal expression of FSH when exposed to Dox, but to be regulated by all the hormones that normally control FSH production in vivo. Feeding maximally effective levels of Dox increased overall mRNA for FSHB and serum FSH by over half in males, and Dox treatment more than doubled the normal ovulation rate of female mice for up to 10 reproductive cycles. Lower levels of Dox increased the number of developing embryos by 30%. Ovarian structure and function appeared normal. In summary, gene switch technology and normal FSH regulation were combined to effectively enhance ovulation in mice. Theoretically, the same strategy can be used with any genetic switch to increase ovulation (or any highly conserved physiology) in any mammal.     

B cell depletion inhibits spontaneous autoimmune thyroiditis in NOD.H-2h4 mice

B cells are important for the development of most autoimmune diseases. B cell depletion immunotherapy has emerged as an effective treatment for several human autoimmune diseases, although it is unclear whether B cells are necessary for disease induction, autoantibody production, or disease progression. To address the role of B cells in a murine model of spontaneous autoimmune thyroiditis (SAT), B cells were depleted from adult NOD.H-2h4 mice using anti-mouse CD20 mAb. Anti-CD20 depleted most B cells in peripheral blood and cervical lymph nodes and 50-80% of splenic B cells. Flow cytometry analysis showed that marginal zone B cells in the spleen were relatively resistant to depletion by anti-CD20, whereas most follicular and transitional (T2) B cells were depleted after anti-CD20 treatment. When anti-CD20 was administered before development of SAT, development of SAT and anti-mouse thyroglobulin autoantibody responses were reduced. Anti-CD20 also reduced SAT severity and inhibited further increases in anti-mouse thyroglobulin autoantibodies when administered to mice that already had autoantibodies and thyroid inflammation. The results suggest that B cells are necessary for initiation as well as progression or maintenance of SAT in NOD.H-2h4 mice.     

p-Chloroamphetamine formation responsible for long-term depletion of brain serotonin after N-cyclopropyl-p-chloroamphetamine injection in rats

After the injection of N-cyclopropyl-p-chloroamphetamine (N-cyclopropyl-PCA) into rats, p-chloroamphetamine (PCA) was identified in brain by high performance liquid chromatography with UV detection and was quantitated by that method and by spectrofluorometric analysis involving reaction with fluorescamine. The identity of PCA in brains of rats treated with N-cyclopropyl-PCA was confirmed by mass spectrometry. The peak concentrations of PCA in brain occurred 4 hrs after N-cyclopropyl-PCA injection. Brain concentrations of PCA and of N-cyclopropyl-PCA were measured at 1 or 4 hrs, respectively, after the injection of various doses of PCA or of N-cyclopropyl-PCA into rats. The depletion of brain serotonin and 5-hydroxyindoleacetic acid (5-HIAA) was measured 1 week after injection of those same doses of PCA or N-cyclopropyl-PCA. Comparing peak concentrations of PCA with the degree of depletion of brain serotonin supported the interpretation that PCA formed metabolically accounted for the long-term depletion of brain 5-hydroxyindoles after injection of N-cyclopropyl-PCA in rats.     

Zinc supplementation inhibits hepatic apoptosis in mice subjected to a long-term ethanol exposure

Hepatocyte apoptosis has been documented in both clinical and experimental alcoholic liver disease. This study was undertaken to examine the effect of dietary zinc supplementation on hepatic apoptosis in mice subjected to a long-term ethanol exposure. Male adult 129S6 mice fed an ethanol-containing liquid diet for 6 months developed hepatitis, as indicated by neutrophil infiltration and elevation of hepatic keratinocyte chemoattractant (KC) and monocyte chemoattractant protein-1 (MCP-1) levels. Apoptotic cell death was detected in ethanol-exposed mice by a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and was confirmed by the increased activities of caspase-3 and -8. Zinc supplementation attenuated alcoholic hepatitis and reduced the number of TUNEL-positive cells in association with inhibition of caspase activities. Ethanol exposure caused oxidative stress, as indicated by reactive oxygen species accumulation, mitochondrial glutathione depletion, and decreased metallothionein levels in the liver, which were suppressed by zinc supplementation. The mRNA levels of tumor necrosis factor (TNF)-alpha, TNF-R1, FasL, Fas, Fas-associated factor-1, and caspase-3 in the liver were upregulated by ethanol exposure, which were attenuated by zinc supplementation. Zinc supplementation also prevented ethanol-elevated serum and hepatic TNF-alpha levels and TNF-R1 and Fas proteins in the liver. In conclusion, zinc supplementation prevented hepatocyte apoptosis in mice subjected to long-term ethanol exposure, and the action of zinc is likely through suppression of oxidative stress and death receptor-mediated pathways.