Intrahippocampal Injection of Endothelin-1: A New Model of Ischemia-induced Seizures in Immature Rats

Summary:  The goal of this study was to develop a new model of ischemia-induced seizures in immature rats using injection of vasoconstrictor Endothelin-1 (ET-1) into the brain. ET-1 (10, 20, or 40 pmol) was infused into the left dorsal hippocampus of freely moving Wistar rats 12 (P12) and 25 (P25) days old. Animals were then video/EEG-monitored for 100 min and monitoring was repeated 22 h later. Parameters of electrographic seizures (frequency and mean duration) as well as pattern of their behavioral correlates were evaluated. The pattern of behavioral seizures was used to develop model-specific scoring system. Cresyl violet and Fluoro Jade-B-staining were used to evaluate brain damage. Extension of the lesion was correlated with seizure severity. After ET-1-injection, seizures occurred in 83–100% animals of all age-and-dose groups and persisted for 24 h except P12 rats with 10 pmol. There were no differences in average seizure duration (18–40 s) or seizure frequency (3–7 seizures/100 min) among individual dose-groups. Between the 1st and 2nd observation period, total seizure duration decreased in 71% of P12 and 47% of P25 rats. Electrographic seizure activity was most frequently accompanied by clonus, incidence of more severe convulsions (barrel rolling or generalized clonic seizures) increased with dose of ET-1. Morphologic examination did not reveal any dose-related difference in damage severity, hippocampal damage was however more extensive in P12 compared to P25 animals. Seizure severity correlated positively with severity of the damage in both age groups. Our study presents focal injection of ET-1 into the brain as a new and practical model of ischemia-induced seizures in immature rats.     

PSD-95 in CA1 Area Regulates Spatial Choice Depending on Age

Cognitive processes that require spatial information rely on synaptic plasticity in the dorsal CA1 area (dCA1) of the hippocampus. Since the function of the hippocampus is impaired in aged individuals, it remains unknown how aged animals make spatial choices. Here, we used IntelliCage to study behavioral processes that support spatial choices of aged female mice living in a group. As a proxy of training-induced synaptic plasticity, we analyzed the morphology of dendritic spines and the expression of a synaptic scaffold protein, PSD-95. We observed that spatial choice training in young adult mice induced correlated shrinkage of dendritic spines and downregulation of PSD-95 in dCA1. Moreover, long-term depletion of PSD-95 by shRNA in dCA1 limited correct choices to a reward corner, while reward preference was intact. In contrast, old mice used behavioral strategies characterized by an increased tendency for perseverative visits and social interactions. This strategy resulted in a robust preference for the reward corner during the spatial choice task. Moreover, training decreased the correlation between PSD-95 expression and the size of dendritic spines. Furthermore, PSD-95 depletion did not impair place choice or reward preference in old mice. Thus, our data indicate that while young mice require PSD-95-dependent synaptic plasticity in dCA1 to make correct spatial choices, old animals observe cage mates and stick to a preferred corner to seek the reward. This strategy is resistant to the depletion of PSD-95 in the CA1 area. Overall, our study demonstrates that aged mice combine alternative behavioral and molecular strategies to approach and consume rewards in a complex environment.SIGNIFICANCE STATEMENT It remains poorly understood how aging affects behavioral and molecular processes that support cognitive functions. It is, however, essential to understand these processes to develop therapeutic interventions that support successful cognitive aging. Our data indicate that while young mice require PSD-95-dependent synaptic plasticity in dCA1 to make correct spatial choices (i.e., choices that require spatial information), old animals observe cage mates and stick to a preferred corner to seek the reward. This strategy is resistant to the depletion of PSD-95 in the CA1 area. Overall, our study demonstrates that aged mice combine alternative behavioral and molecular strategies to approach and consume rewards in a complex environment. Second, the contribution of PSD-95-dependent synaptic functions in spatial choice changes with age.     

The relationship between microstructure and photocatalytic behavior in lanthanum-modified 2D TiO2 nanosheets upon annealing of a freeze-cast precursor

Titanium dioxide modified with 3 wt% La was prepared via a green freeze-casting method, and its photocatalytic activity was tested in terms of its ability to degrade 4-chlorophenol (4-CP) and remove total organic carbon (TOC). Under annealing conditions, the freeze-cast precursor was transformed into an La-modified anatase with a well-defined 2D TiO₂ nanosheet morphology. Rietveld refinement of the X-ray diffraction patterns confirmed the substitutional nature of the La cation that induced local structural variations and involved subtle ion displacement in the TiO₂ lattice due to the ionic size effect. Despite nearly identical tetragonal structures, replacement of Ti with La alters the photocatalytic activity through a reduction in band gap energies and an increase in charge carrier mobility. Material annealed at 650 °C exhibited the highest photocatalytic performance and achieved efficient TOC removal. Upon annealing at 800 °C, nanoscale lanthanum-enriched regions were generated due to the diffusive migration of La cations and phase transition from anatase to rutile. The La³⁺ cation, acting as a structural promoter, supported 2D TiO₂ growth with well controlled crystallite size, surface area and porosity. La³⁺ could be regarded as a potential electronic promoter that can reduce the band gap of 2D TiO₂ nanosheets and can provide a signature of the electron transfer and carrier charge separation. Both methods, kinetics of degradation of 4-CP and TOC, provided similar results, revealing that the photocatalytic activity under UV light irradiation increased in the order 950C < 500 °C < 800 °C < 650 °C < TiO₂-P25.

Titanium dioxide modified with 3 wt% La was prepared via a green freeze-casting method, and its photocatalytic activity was tested in terms of its ability to degrade 4-chlorophenol (4-CP) and remove total organic carbon (TOC).     

Plasma protein haptoglobin modulates renal iron loading

Haptoglobin is the plasma protein with the highest binding affinity for hemoglobin. The strength of hemoglobin binding and the existence of a specific receptor for the haptoglobin-hemoglobin complex in the monocyte/macrophage system clearly suggest that haptoglobin may have a crucial role in heme-iron recovery. We used haptoglobin-null mice to evaluate the impact of haptoglobin gene inactivation on iron metabolism. Haptoglobin deficiency led to increased deposition of hemoglobin in proximal tubules of the kidney instead of the liver and the spleen as occurred in wild-type mice. This difference in organ distribution of hemoglobin in haptoglobin-deficient mice resulted in abnormal iron deposits in proximal tubules during aging. Moreover, iron also accumulated in proximal tubules after renal ischemia-reperfusion injury or after an acute plasma heme-protein overload caused by muscle injury, without affecting morphological and functional parameters of renal damage. These data demonstrate that haptoglobin crucially prevents glomerular filtration of hemoglobin and, consequently, renal iron loading during aging and following acute plasma heme-protein overload.     

TLR3-mediated IFN-β gene induction is negatively regulated by the TLR adaptor MyD88 adaptor-like

There is limited insight into the mechanisms involved in the counterregulation of TLR. Given the important role of TLR3/TIR domain-containing adaptor-inducing IFN-β (TRIF)-dependent signalling in innate immunity, novel insights into its modulation is of significance in the context of many physiological and pathological processes. Herein, we sought to perform analysis to definitively assign a mechanistic role for MyD88 adaptor-like (Mal), an activator of TLR2/4 signalling, in the negative regulation of TLR3/TRIF signalling. Biochemical and functional analysis demonstrates that Mal negatively regulates TLR3, but not TLR4, mediated IFN-β production. Co-immunoprecipitation experiments demonstrate that Mal associates with IRF7 (IRF, IFN regulatory factor), not IRF3, and Mal specifically blocks IRF7 activation. In doing so, Mal impedes TLR3 ligand-induced IFN-β induction. Interestingly, Mal does not affect the induction of IL-6 and TNF-α upon TLR3 ligand engagement. Together, these data show that the TLR adaptor Mal interacts with IRF7 and, in doing so, impairs IFN-β induction through the positive regulatory domains I-III enhancer element of the IFN-β gene following poly(I:C) stimulation. Our findings offer a new mechanistic insight into TLR3/TRIF signalling through a hitherto unknown mechanism whereby Mal inhibits poly(I:C)-induced IRF7 activation and concomitant IFN-β production. Thus, Mal is essential in restricting TLR3 signalling thereby protecting the host from unwanted immunopathologies associated with excessive IFN-β production.     

Discovery of hundreds of mirtrons in mouse and human small RNA data

Atypical miRNA substrates do not fit criteria often used to annotate canonical miRNAs, and can escape the notice of miRNA genefinders. Recent analyses expanded the catalogs of invertebrate splicing-derived miRNAs ("mirtrons"), but only a few tens of mammalian mirtrons have been recognized to date. We performed meta-analysis of 737 mouse and human small RNA data sets comprising 2.83 billion raw reads. Using strict and conservative criteria, we provide confident annotation for 237 mouse and 240 human splicing-derived miRNAs, the vast majority of which are novel genes. These comprise three classes of splicing-derived miRNAs in mammals: conventional mirtrons, 5'-tailed mirtrons, and 3'-tailed mirtrons. In addition, we segregated several hundred additional human and mouse loci with candidate (and often compelling) evidence. Most of these loci arose relatively recently in their respective lineages. Nevertheless, some members in each of the three mirtron classes are conserved, indicating their incorporation into beneficial regulatory networks. We also provide the first Northern validation for mammalian mirtrons, and demonstrate Dicer-dependent association of mature miRNAs from all three classes of mirtrons with Ago2. The recognition of hundreds of mammalian mirtrons provides a new foundation for understanding the scope and evolutionary dynamics of Dicer substrates in mammals.