Misoprostol attenuates neonatal cardiomyocyte proliferation through Bnip3, perinuclear calcium signaling, and inhibition of glycolysis.

Systemic hypoxia resulting from preterm birth, altered lung development, and cyanotic congenital heart disease is known to impede the regulatory and developmental pathways in the neonatal heart. While the molecular mechanisms are still unknown, hypoxia induces aberrant cardiomyocyte proliferation, which may be initially adaptive, but can ultimately program the heart to fail in early life. Recent evidence suggests that the prostaglandin E1 analogue, misoprostol, is cytoprotective in the hypoxia-exposed neonatal heart by impacting alternative splicing of the Bcl-2 family member Bnip3, resulting in the generation of a variant lacking the third exon (Bnip3ΔExon3 or small Nip; sNip). Using a rodent model of neonatal hypoxia, in combination with rat primary neonatal cardiomyocytes (PVNCs) and H9c2 cells, we sought to determine if misoprostol can prevent cardiomyocyte proliferation and what the key molecular mechanisms might be in this pathway. In PVNCs, exposure to 10% oxygen induced myocyte proliferation concurrent with molecular markers of cell-cycle progression, such as Cyclin-D1, which were prevented by misoprostol treatment. Furthermore, we describe a critical role for sNip in opposing cardiomyocyte proliferation through several mechanisms, including reduced expression of the proliferative MEF2C-myocardin-BMP10 pathway, accumulation of nuclear calcium leading to NFATc3 activation, and increased expression of the cardiac maturation factor BMP2. Intriguingly, misoprostol and sNip inhibited hypoxia-induced glycolytic flux, which directly influenced myocyte proliferation. These observations were further supported by knockdown studies, where hypoxia-induced cardiomyocyte proliferation is restored in misoprostol-treated cells by an siRNA targeting sNip. Finally, in postnatal day (PND)-10 rat pups exposed to hypoxia, we observed histological evidence of increased nuclei number and increased PPH3 staining, which were completely attenuated by misoprostol treatment. Collectively, this data demonstrates how neonatal cardiomyocyte proliferation can be pharmacologically modulated by misoprostol treatment, which may have important implications for both neonatal and regenerative medicine.

Acute ß-tetrabromoethylcyclohexane (ß-TBECH) treatment inhibits the electrical activity of rat Purkinje neurons.

Brominated flame-retardants are environmentally pervasive and persistent synthetic chemicals, some of which have been demonstrated to disrupt neuroendocrine signaling and electrical activity of neurons. 1,2-dibromo-4-(1,2-dibromoethyl)-cyclohexane (TBECH) lacks the toxicity of other classes of BFRs, however its safety is still questioned, as little is known of its neurological effects. Therefore, we sought to determine if TBECH could acutely alter the electrical activity of Purkinje neurons maintained in vitro. Briefly, cerebella from gestational day 20 rats were dissociated and maintained for up to three weeks in culture. Action potentials of Purkinje neurons were detected by cell-attached patch clamp before, during, and after application of ß-TBECH. ß-TBECH decreased action potential activity in a dose-dependent manner with an apparent EC50 of 396 nM. ß-TBECH did not significantly alter the coefficient of variation, a measure of the regularity of firing, suggesting that the mechanism of ß-TBECH's effects on firing frequency may be independent of Purkinje neuron intracellular calcium handling. Because levels of ß-TBECH in exposed individuals may not approach the EC50, these data suggest that any abnormal neurodevelopment or behavior linked with ß-TBECH exposure may result from endocrinological effects as opposed to direct disruption of electrical activity.

Misoprostol regulates Bnip3 repression and alternative splicing to control cellular calcium homeostasis during hypoxic stress.

The cellular response to hypoxia involves the activation of a conserved pathway for gene expression regulated by the transcription factor complex called hypoxia-inducible factor (HIF). This pathway has been implicated in both the adaptive response to hypoxia and in several hypoxic-ischemic-related pathologies. Perinatal hypoxic injury, often associated with prematurity, leads to multi-organ dysfunction resulting in significant morbidity and mortality. Using a rodent model of neonatal hypoxia and several representative cell lines, we observed HIF1α activation and down-stream induction of the cell death gene Bnip3 in brain, large intestine, and heart which was mitigated by administration of the prostaglandin E1 analog misoprostol. Mechanistically, we determined that misoprostol inhibits full-length Bnip3 (Bnip3-FL) expression through PKA-mediated NF-κB (P65) nuclear retention, and the induction of pro-survival splice variants. We observed that the dominant small pro-survival variant of Bnip3 in mouse cells lacks the third exon (Bnip3ΔExon3), whereas human cells produce a pro-survival BNIP3 variant lacking exon 2 (BNIP3ΔExon2). In addition, these small Bnip3 splice variants prevent mitochondrial dysfunction, permeability transition, and necrosis triggered by Bnip3-FL by blocking calcium transfer from the sarco/endoplasmic reticulum to the mitochondria. Furthermore, misoprostol and Bnip3ΔExon3 promote nuclear calcium accumulation, resulting in HDAC5 nuclear export, NFAT activation, and adaptive changes in cell morphology and gene expression. Collectively, our data suggests that misoprostol can mitigate the potential damaging effects of hypoxia on multiple cell types by activating adaptive cell survival pathways through Bnip3 repression and alternative splicing.

BDNF expression increases without changes in play behavior following concussion in juvenile rats (Rattus Norvegicus).

PURPOSE: Young children have a high risk of concussion or mild traumatic brain injury (mTBI). Children often appear healthy soon after mTBI, but some have pervasive cognitive and/or motor impairments. Understanding underlying mechanisms recruited after concussion may help for return to play protocols and mitigating what might be lifelong impairments. METHODS: We investigated molecular and behavioral changes in a rat model of childhood concussion. Rats received an injury or sham procedure at an age approximately equivalent to the human period of early childhood. Social play was analyzed for behavioral differences. Tissue from the right motor cortex (impacted), left motor cortex, and medial prefrontal cortex were analyzed for brain derived neurotrophic factor (BDNF) protein. RESULTS: Play behavior was not significantly different between conditions. BDNF levels were much higher in both the right and left motor cortices of the mTBI group compared to medial prefrontal cortex, which is relatively remote from the impact site, within the mTBI group and all tissue collected from the sham group. CONCLUSIONS: There is ongoing plastic change at the cellular level in both the impacted area and the well-connected contralateral area after a concussion, suggesting compensatory mechanisms after injury are still at play.

Protection from Psoriasis-Related Thrombosis after Inhibition of IL-23 or IL-17A.

Psoriasis patients experience chronic systemic skin inflammation and develop cardiovascular comorbidities that shorten their lifespan. Whether cardiovascular disease is improved by treatment with current biologics that target disease-specific pathways is unclear. KC-Tie2 mice develop psoriasiform skin inflammation with increases in IL-23 and IL-17A and proinflammatory monocytosis and neutrophilia that precedes development of carotid artery thrombus formation. To examine whether targeted blockade of IL-23 or IL-17A in KC-Tie2 psoriasis mice improves cardiovascular outcomes, mice were treated systemically for 6 weeks with antibodies targeting IL-17A, IL-17RA, IL-12/23p40, or IL-23p19. Skin inflammation; thrombosis clotting times; and percentage of splenic monocytes, neutrophils, and CD4 T cells were examined. Skin inflammation significantly improved in KC-Tie2 mice treated with each of the antibodies targeting IL-23, IL-17A, or IL-17RA, consistent with clinical efficacy observed in psoriasis patients. The time to occlusive thrombus formation lengthened in these mice and correlated with attenuated acanthosis. This decrease in skin inflammation paralleled decreases in splenic neutrophils (CD11b+Ly6G+) but not monocytes (CD11b+Ly6Chigh) or T cells (CD4+). Our data show that targeted inhibition of IL-23 or IL-17A improves psoriasis-like skin disease and also improves cardiovascular disease in mice.

Experience with featural-cue reliability influences featural- and geometric-cue use by mice (Mus musculus).

Orienting is a critical skill for all mobile animals. Two commonly studied visual components used to guide orientation in an environment are geometric (e.g., distance or direction) and featural cues (e.g., color or texture). Previous research has shown that visual-cue use and cue weighing can depend on the navigator's previous experience, the nature and reliability of the cues, and genetic factors. Accordingly, the domestic mouse (Mus musculus) is a species of increasing interest because of its potential as a model for human neurological disorders with associated spatial disorientation, as is seen in Alzheimer's disease. In the present study, adult C57BL/6 mice were trained to search for a hidden food reward in one corner of a rectangular environment with featural information displayed continuously along the walls. After training, one group of mice was given a block of testing in which the featural information was removed, followed by a second block of testing in which the featural information was put in conflict with the learned configuration of featural and geometric cues. A second group of mice was given the same set of tests, but in the reverse order. Our results show that the mice incidentally encoded the geometry of the environment if they had experience with featural cues being unreliable prior to tests, during which featural cues were completely removed (unstable). Furthermore, we found when featural and geometric cues provide conflicting spatial information, this unreliability of featural cues over the course of the study may influence cue weighing. (PsycINFO Database Record

Changes in dendritic morphology and spine density in motor cortex of the adult rat after stroke during infancy.

Cognitive and motor deficits are pervasive in children that suffer early brain injury. The aim of this study was to determine the impact that early damage has on dendritic spine density and other aspects of dendritic morphology of neurons in the motor cortex. Also of interest was how changes in dendritic structure evolved across the lifespan. Ischemia was induced in 10-day-old Long Evans rats by injection of Rose Bengal dye and a laser positioned over right motor cortex. Animals were sacrificed at two and six months of age, and brains were processed for Golgi-Cox staining. Animals exposed to early damage exhibited increases in length of basilar dendrites at two months of age, however no differences in spine density were found across groups at this age. At six months of age, injured animals demonstrated an overall decrease in apical and basilar spine density. Our results suggest that the changes in dendritic length and spine density observed after early damage are unable to be maintained as the animal ages. The observation that increases in spine density do not necessarily coincide with increases in dendritic length suggests that the two processes may not be dependent on one another and suggest two independent plasticity processes responding to damage.

Altered morphology of motor cortex neurons in the VPA rat model of autism.

Autism occurs in 1 in 1,000 children and incidence may be increasing. Investigating brain development and developmental injury in humans is difficult. As such, many studies rely on animal models of disorders. We chose to investigate the valproic acid-exposed rat, as this model shares many similarities with autism. Pregnant Long-Evans rats were administered either valproic acid (VPA) or saline during fetal neural tube development. Morphological analyses of cells in layer II of the golgi impregnated motor cortex were done to determine dendritic length, volume, and complexity in both groups. No differences were found in length or volume of cortical dendrites, but dendritic arborization was more complex in apical dendrites of pyramidal cells in VPA-exposed animals than controls. The implication of this finding is that pruning in the VPA-exposed rat is not occurring, which is consistent with theories related to abnormal human brain development in autism.

Acute administration of interleukin-1beta disrupts motor learning.

Proinflammatory cytokines have been shown to disrupt the normal transfer of short-term memory to long-term storage sites. Previous research has focused predominantly on the effect of cytokines on hippocampus-mediated spatial learning. To further understand the effects of cytokines on learning and memory, the authors evaluated the effects of interleukin-1beta (IL-1beta) on a motor learning task. Male Long-Evans rats were rewarded with food pellets after they traversed a runway. The runway was either flat (control condition) or had up-ended dowels (motor learning condition). Subjects traversed the flat runway or dowel task for 5 days, 10 trials per day, and were treated with either saline or with 4 microg/kg IL-1beta immediately after training on the first 2 days. Rats in the motor learning task treated with IL-1beta were consistently slower at traversing the runway. IL-1beta did not impair performance in the control condition; rats in the flat condition performed similarly regardless of whether they were treated with saline or IL-1beta. These data are the first evidence demonstrating IL-1beta can disrupt performance in a motor learning task.

Cerebral angiogenic factors, angiogenesis, and physiological response to chronic hypoxia differ among four commonly used mouse strains.

Angiogenesis is a critical element for adaptation to low levels of oxygen and occurs following long-term exposure to mild hypoxia in rats. To test whether a similar response in mice occurs, CD1, 129/Sv, C57Bl/6, and Balb/c mice were exposed to 10% oxygen for up to 3 wk. All mice showed significant increases in the percentage of packed red blood cells, and CD1 and 129/Sv mice showed increased respiration frequency and minute volume, common physiological measures of hypoxia. Significant angiogenesis was observed in all strains except Balb/c following 3-wk exposure to chronic hypoxia. CD1 hypoxic mice had the largest increase (88%), followed by C57Bl/6 (48%), 129/Sv (41%), and Balb/c (12%), suggesting that some mice undergo more remodeling than others in response to hypoxia. Protein expression analysis of vascular endothelial growth factor (VEGF), angiopoietin (Ang)-1 and Ang2, and Tie2 were examined to determine whether regulation of different angiogenic proteins could account for the differences observed in hypoxia-induced angiogenesis. CD1 mice showed the strongest upregulation of VEGF, Ang2, Ang1, and Tie2, whereas Balb/c had only subtle increases in VEGF and no change in the other proteins. C57Bl/6 mice showed a regulatory response that fell between the CD1 and Balb/c mice, consistent with the intermediate increase in angiogenesis. Our results suggest that genetic heterogeneity plays a role in angiogenesis and regulation of angiogenic proteins and needs to be accounted for when designing and interpreting experiments using transgenic mice and when studying in vivo models of angiogenesis.

MAP2 and synaptophysin protein expression following motor learning suggests dynamic regulation and distinct alterations coinciding with synaptogenesis.

Learning a new motor skill can induce neuronal plasticity in rats. Within motor cortex, learning-induced plasticity includes dendritic reorganization, synaptogenesis, and changes in synapse morphology. Behavioral studies have demonstrated that learning requires protein synthesis. It is likely that some of the proteins synthesized during learning are involved in, or the result of, learning-induced structural plasticity. We predicted the expression of proteins involved in neural plasticity would be altered in a learning dependent fashion. Long-Evans rats were trained on a series of motor tasks that varied in complexity, so that the effects of activity could be teased apart from the effects of learning. The motor cortices were examined for MAP2 and synaptophysin protein using Western blotting and immunohistochemistry. Western blotting revealed that expression of MAP2 was not detectably influenced by learning, whereas synaptophysin expression increased on day 1, 3, and 5 of complex motor skill learning. Expression of MAP2 does not seem to indicate difficulty of task or duration of training time, whereas increases in synaptophysin expression, which appear diffusely across the cortex, seem to be correlated with the first 5 days of motor skill learning. Similar findings with GAP-43 suggest the change in synaptophysin may coincide with synapse formation. Immunohistochemistry did not reveal any localized changes in protein expression. These data indicate a difference in learning-induced expression in the mammalian brain compared to reports in the literature, which have often focused on stimulation to induce alterations in protein expression.

Persistent motor deficit following infusion of autologous blood into the periventricular region of neonatal rats.

Periventricular hemorrhage (PVH) in the brain of premature infants is often associated with developmental delay and persistent motor deficits. Our goal is to develop a rodent model that mimics the behavioral phenotype. We hypothesized that autologous blood infusion into the periventricular germinal matrix region of neonatal rats would lead to immediate and long-term behavioral changes. Tail blood or saline was infused into the periventricular region of 1-day-old rats. Magnetic resonance (MR) imaging was used to demonstrate the hematoma. Rats with blood infusion, as well as saline and intact controls, underwent behavior tests until 10 weeks age. Blood-infused rats displayed significant delay in motor development (ambulation, righting response, and negative geotaxis) to 22 days of age. As young adults, they exhibited impaired ability to stay on a rotating rod and to reach for food pellets. MR imaging at 10 weeks demonstrated subsets of rats with normal appearing brains, focal cortical infarcts, or mild hydrocephalus. There was a good correlation between MR imaging and histological findings. Some rats exhibited periventricular heterotopia and/or subtle striatal abnormalities not apparent on MR images. We conclude that autologous blood infusion into the brain of neonatal rats successfully models some aspects of periventricular hemorrhage that occurs after premature birth in humans.

Vascular-specific growth factor angiopoietin 1 is involved in the organization of neuronal processes.

Neuronal processes and vessels have similar branching and bifurcation patterning in the adult body and appear to use many of the same molecules during their development, including vascular endothelial growth factor, Notch, neuropilin, and ephrins/Ephs. We were interested in determining whether the endothelial growth factor angiopoietin (Ang) has a unique role in the nervous system in addition to its angiogenic role. By using a mouse molecular genetics approach, we overexpressed Ang1 in the mouse forebrain and observed increases in overall vascularization, consistent with prior reports describing the role of Ang1. Nonvascular events, involving alterations in the dendritic organization of layer II motor cortex neurons, dentate granule cells, and pyramidal cells of CA1, were seen, suggesting that Ang1 was able to influence the growth of these processes. The angiopoietin tyrosine kinase receptor Tie2 was not found on neurons or their processes, but beta1 integrin was and has previously been found to act as an Ang receptor. Our findings provide some of the first data evaluating the interactions between the developing nervous system and the vascular protein Ang1. Understanding interactions between the developing nervous and vascular systems will lead to novel insight into how the two systems interact throughout development, during senescence, and in disease.

Anesthetized Long Evans rats show similar protein expression and long-term potentiation as Fischer 344 rats but reduced short-term potentiation in motor cortex.

A number of studies describe strain-related differences in the motor behavior of rats. Inbred albino F344 rats are found to be impaired in procedural spatial learning, skilled reaching, and over ground locomotion in relation to pigmented out bred Long Evans (LE) rats. These deficits could be related to the functional differences in the motor cortex of the two strains, and the objective of the present study was to examine this hypothesis. Synaptic transmission was examined in the two rat strains, using long-term potentiation (LTP) and short-term potentiation (STP), two electrophysiological measures of neural function and learning. Field potentials were evoked in the motor cortex of anesthetized Long Evans and Fischer 344 (F344) rats in response to contralateral white matter stimulation. The main findings indicated that (1) baseline-evoked responses in the two strains was similar, indicating similar basal levels of synaptic strength, (2) LTP was induced in both strains of rats, suggesting similar synaptic efficacy in the two strains of rats, and (3) STP was enhanced in the Fischer 344 rats, suggesting differences in synaptic function. Protein expression also revealed that the two strains did not differ with respect to structural or synaptic protein expression. Thus, the two strains exhibit motor skill differences despite a great degree of physiological similarity in motor cortex. The results are discussed in relation to the greater utility of using the Long Evans rat for examining the neural basis of plasticity and models of disease, especially if motor tasks are evaluated.

Unique changes in synaptic morphology following tetanization under pharmacological blockade.

Long-term potentiation (LTP) in the hippocampus has been associated with changes in synaptic morphology. Whether these changes are LTP-dependent or simply a result of electrophysiological stimulation has not yet been fully determined. This study involved an examination of synaptic morphology in the rat dentate gyrus 24 h after electrophysiological stimulation sufficient to induce LTP. In one group, ketamine, a competitive NMDA antagonist, was injected prior to stimulation to block the formation of LTP. Synaptic morphological quantification included estimating the total number of synapses per neuron, determining synaptic curvature and the presence of synaptic perforations, and measuring the maximal PSD profile length of the synapses. The results indicated that most of the changes observed following the induction of LTP (increases in the proportion of concave-shaped synapses, increases in perforated concave synapses, and a decrease in the length of nonperforated concave synapses) are not observed under ketamine blockade, suggesting that they are LTP-specific and not simply the result of tetanic stimulation. Ketamine was associated, however, with several novel structural changes including a decrease in the length of the perforations in the concave perforated synapses, a reduction in the number of convex perforated synapses, and a nonlayer-specific increase in synaptic length compared to controls. Based on previous research, this combination of morphological characteristics is potentially less efficacious, which suggests that synapses that are tetanized but not potentiated, due to pharmacological blockade, appear to undergo opposing, compensatory, or homeostatic changes. These results support the suggestion that synaptic morphology changes are both stimulation- and area-specific, are highly complex, and depend on the specific local physiology.

Altered mossy fiber distributions in adult Fmr1 (FVB) knockout mice.

The fragile-X mental retardation protein (FMRP) is greatly reduced or absent in individuals with fragile-X mental retardation syndrome, a common, heritable form of mental retardation. Morphological studies suggest that this protein functions in normal synapse maturation and neuronal plasticity. Examination of human brain autopsy tissue has shown that fragile-X patients exhibit long, thin spines more frequently, and stubby mushroom-shaped spines less frequently, than these two types of spines are seen in normal autopsy tissue. Fragile-X tissue also has a greater density of these spines along dendrites, which suggests a possible failure of synapse elimination. Fmr1 knockout mice and wild-type littermates brains were processed for Timm staining, which reveals the zinc-rich terminals of the dentate gyrus, the mossy fibers. The Fmr1 knockout mice exhibited a pattern of Timm granule-staining within the stratum oriens of subfield CA3 and the inner molecular layer that was significantly different than staining seen in wild-type animals. The sources and consequences of the altered terminal staining are unclear, but are discussed in relation to immature synapse morphology, a failure of normal regression of synapses, and a potential biological penalty of such a failure to regress.