evaluation-blood-brain-barrier-breakdown-mouse-model-mild-traumatic

Evaluation of Blood-Brain Barrier Breakdown in a Mouse Model of Mild Traumatic Brain Injury

Fluorescent dyes are used to determine the extent of dye extravasation that occurs due to blood-brain barrier (BBB) breakdown. Labeling with these dyes is ...

National Defense Medical College

Intercellular communication via gap junctions allows cells within multicellular organisms to share small molecules. The effect of such interactions has been elucidated using mouse gene knockout strategies. Although several mutations in human gap junction-encoding connexin (Cx) have been described, Cx mutants in mice do not always recapitulate the human disease. Among the 20 mouse Cxs, Cx26, Cx43, and Cx45 play roles in early cardiac or placental development, and disruption of the genes results in lethality that hampers further analyses. Embryonic stem cells (ESCs) that lack Cx43 or Cx45 have made analysis feasible in both in vitro differentiated cell cultures and in vivo chimeric tissues. The success of mouse ESCs studies is leading to the use of induced pluripotent stem cells to learn more about the pathogenesis of human Cx diseases. This review summarizes the current status of mouse Cx disruption models and ESC differentiation studies, and discusses their implication for understanding human Cx diseases.

Connexin mutant embryonic stem cells and human diseases.

Epigenetic factors of pressure load, fluid force, and electrical fields that occur during cardiac contraction affect cardiac development, morphology, function, and pathogenesis. These factors are orchestrated by intercellular communication mediated by gap junctions, which synchronize action potentials and second messengers. Misregulation of the gap junction protein connexin (Cx) alters cardiogenesis, and can be a pathogenic factor causing cardiac conduction disturbance, fatal arrhythmia, and cardiac remodeling in disease states such as hypertension and ischemia. Changes in Cx expression can occur even when the DNA sequence of the Cx gene itself is unaltered. Posttranslational modifications might reduce arrhythmogenic substrates, improve cardiac function, and promote remodeling in a diseased heart. In this review, we discuss the epigenetic features of gap junctions that regulate cardiac morphology and remodeling. We further discuss potential clinical applications of current knowledge of the structure and function of gap junctions.

Gap junctional regulation of pressure, fluid force, and electrical fields in the epigenetics of cardiac morphogenesis and remodeling.

Among gap junction-encoding genes, the loss of connexin (Cx) 45 most profoundly obstructs embryogenesis through an endocardial cushion defect and conduction block. However, the interdependence of these defects is not known, and the details of conduction block have not been elucidated. Here, we examined mouse embryos with a region-specific deletion of Cx45 in the myocardium (CA-Cre; Cx45(flox/flox)) or endothelium (Tie2-Cre; Cx45(flox/flox)). Although the deletion of Cx45 in the myocardium was heterogeneous, the CA-Cre; Cx45(flox/flox) embryos were lethal at the same stage as the constitutive Cx45-deficient (Cx45(-/-)) embryos. We determined the onset and patterns of their conduction block through point-tracking in video recordings of embryonic heart contractions. An incomplete conduction block at the atrioventricular canal appeared at embryonic day (E) 8.5 and was predominant around the lethal E9.5 stage in both the Cx45(-/-) and CA-Cre; Cx45(flox/flox) embryos. Although the Cx45(-/-) hearts showed a consistently severe reduction in atrioventricular conduction velocity, the CA-Cre; Cx45(flox/flox) hearts had delay times within the normal range and showed frequent retrograde conduction. As previously reported, the Cx45(-/-) endocardial cushion was consistently defective, and nuclear factor of activated T-cells cytoplasmic (NFATc)1 within the endocardium showed inactive cytoplasmic distribution. In CA-Cre; Cx45(flox/flox), however, the endocardial cushion was partially formed, with active NFATc1 within the endocardium. There was no developmental abnormality in the Tie2-Cre; Cx45(flox/flox) embryos. These results indicate that myocardial dysfunction is responsible for most of the reported defects in Cx45(-/-), which are alleviated by sporadic Cx45 expression in the CA-Cre; Cx45(flox/flox) myocardium.

Connexin45 contributes to global cardiovascular development by establishing myocardial impulse propagation.

Gap junctional intercellular communication is assumed to play an important role during pre- and peri-implantation development. In this study, we eliminated connexin43 (Cx43) and connexin45 (Cx45), major gap junctional proteins in the pre- and peri-implantation embryo. We generated Cx43 -/- Cx45 -/- embryos by Cx43 +/- Cx45 +/- intercrossing, because mice deficient in Cx43 (Cx43 -/-) exhibit perinatal lethality and those deficient in Cx45 (Cx45 -/-) exhibit early embryonic lethality. Wild-type, Cx43 -/-, Cx45 -/-, and Cx43 -/- Cx45 -/- blastocysts all showed similar outgrowths in in vitro culture. Moreover, Cx43 -/- Cx45 -/- embryos were obtained at the expected Mendelian ratio up to embryonic day 9.5, when the Cx45 -/- mutation proved lethal. The Cx43 -/- Cx45 -/- embryos seemed to have no additional developmental abnormalities in comparison with the single knockout strains. Thus, pre- and peri-implantation development does not require Cx43 and Cx45. Other gap junctional proteins are expressed around these stages and these may compensate for the lack of Cx43 and Cx45.

Absence of connexin43 and connexin45 does not disturb pre- and peri-implantation development.

Inherited cardiac conduction disease is a rare bradyarrhythmia associated with mutations in various genes that affect action potential propagation. It is often characterized by isolated conduction disturbance of the His-Purkinje system, but it is rarely described as a syndromic form.

Progressive Atrial Conduction Defects Associated With Bone Malformation Caused by a Connexin-45 Mutation.

Detonation of explosive devices creates blast waves, which can injure brains even in the absence of external injuries. Among these, blast-induced mild traumatic brain injury (bmTBI) is increasing in military populations, such as in the wars in Afghanistan, Iraq, and Syria. Although the clinical presentation of bmTBI is not precisely defined, it is frequently associated with psycho-neurological deficits and usually manifests in the form of poly-trauma including psychiatric morbidity and cognitive disruption. Although the underlying mechanisms of bmTBI are largely unknown, some studies suggested that bmTBI is associated with blood-brain barrier disruption, oxidative stress, and edema in the brain. The present study investigated the effects of novel antioxidant, molecular hydrogen gas, on bmTBI using a laboratory-scale shock tube model in mice. Hydrogen gas has a strong prospect for clinical use due to easy preparation, low-cost, and no side effects. The administration of hydrogen gas significantly attenuated the behavioral deficits observed in our bmTBI model, suggesting that hydrogen application might be a strong therapeutic method for treatment of bmTBI.

Molecular Hydrogen Prevents Social Deficits and Depression-Like Behaviors Induced by Low-Intensity Blast in Mice.

 Blast-induced shock waves (BSWs) are responsible for several aspects of psychiatric disorders that are collectively termed mild traumatic brain injury (mTBI). The pathophysiology of mTBI includes vascular leakage resulting from blood-brain barrier (BBB) disruption. In this study, the precise sequence of BBB breakdown was examined using an Evans blue and fluorescein isothiocyanate (FITC)-dextran double labeling technique. Evans blue solution was injected into the tail vein of male C57BL6/J mice just before and 4 h, 1 day, 3 days, and 7 days after a single BSW exposure at as low as 25-kPa peak overpressure. In contrast, the FITC-dextran solution was transcardially injected just before perfusion fixation. Differences in the labeling time-point revealed that BBB breakdown was initiated after approximately 3 h, with significant remodeling by 1 day, and continued until 7 days after BSW exposure. BBB breakdown was upregulated in three distinct regions, namely the brain surface and subsurface areas facing the skull, regions closely associated with capillaries, and the circumventricular organ and choroid plexus. These regions showed distinct responses to BSW; moreover, clusters of reactive astrocytes were closely associated with the sites of BBB breakdown. In severe cases, these reactive astrocytes recruited activated microglia. Our findings provide important insights into the pathogenesis underlying mTBI and indicate that even mild BSW exposure affects the whole brain.

Evans Blue and Fluorescein Isothiocyanate-Dextran Double Labeling Reveals Precise Sequence of Vascular Leakage and Glial Responses After Exposure to Mild-Level Blast-Associated Shock Waves.

Article	Year
Connexin mutant embryonic stem cells and human diseases. World journal of stem cells\| PubMed ID: 25426253	2014
Gap junctional regulation of pressure, fluid force, and electrical fields in the epigenetics of cardiac morphogenesis and remodeling. Life sciences\| PubMed ID: 25447447	2015
Connexin45 contributes to global cardiovascular development by establishing myocardial impulse propagation. Mechanisms of development\| PubMed ID: 26916723	2016
Absence of connexin43 and connexin45 does not disturb pre- and peri-implantation development. Zygote (Cambridge, England)\| PubMed ID: 27172058	2016
Progressive Atrial Conduction Defects Associated With Bone Malformation Caused by a Connexin-45 Mutation. Journal of the American College of Cardiology\| PubMed ID: 28705318	2017
Molecular Hydrogen Prevents Social Deficits and Depression-Like Behaviors Induced by Low-Intensity Blast in Mice. Journal of neuropathology and experimental neurology\| PubMed ID: 30053086	2018
Evans Blue and Fluorescein Isothiocyanate-Dextran Double Labeling Reveals Precise Sequence of Vascular Leakage and Glial Responses After Exposure to Mild-Level Blast-Associated Shock Waves. Journal of neurotrauma\| PubMed ID: 36680750	2023

Kiyomasa Nishii

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