Phosphorylation of H2AX is a response to DNA damage, but γH2AX also associates with mitosis and/or apoptosis. We examined the effects of X-rays on DNA integrity to shed more light on the significance ...of H2AX phosphorylation and its relationship with activation of caspase 3 (CASP3), the main apoptotic effector. After administration of the S phase marker BrdU, brains were collected from untreated and irradiated (10 Gray) 24-month-old mice surviving 15 or 30 min after irradiation. After paraffin embedding, brain sections were single- or double-stained with antibodies against γH2AX, p53-binding protein 1 (53BP1) (which is recruited during the DNA damage response (DDR)), active CASP3 (cCASP3), 5-Bromo-2-deoxyuridine (BrdU), and phosphorylated histone H3 (pHH3) (which labels proliferating cells). After statistical analysis, we demonstrated that irradiation not only induced a robust DDR with the appearance of γH2AX and upregulation of 53BP1 but also that cells with damaged DNA attempted to synthesize new genetic material from the rise in BrdU immunostaining, with increased expression of cCASP3. Association of γH2AX, 53BP1, and cCASP3 was also evident in normal nonirradiated mice, where DNA synthesis appeared to be linked to disturbances in DNA repair mechanisms rather than true mitotic activity.
The first description of the
mutation in mouse dates to more than fifty years ago, and later, its causative gene (
) was discovered in mouse, and its human orthologue (
) was demonstrated to be ...causative of lissencephaly 2 (LIS2) and about 20% of the cases of autosomal-dominant lateral temporal epilepsy (ADLTE). In both human and mice, the gene encodes for a glycoprotein referred to as reelin (Reln) that plays a primary function in neuronal migration during development and synaptic stabilization in adulthood. Besides LIS2 and ADLTE,
and/or other genes coding for the proteins of the Reln intracellular cascade have been associated substantially to other conditions such as spinocerebellar ataxia type 7 and 37,
-associated cerebellar hypoplasia,
-associated lissencephaly, autism, and schizophrenia. According to their modalities of inheritances and with significant differences among each other, these neuropsychiatric disorders can be modeled in the homozygous (
) or heterozygous (
)
mouse. The worth of these mice as translational models is discussed, with focus on their construct and face validity. Description of face validity, i.e., the resemblance of phenotypes between the two species, centers onto the histological, neurochemical, and functional observations in the cerebral cortex, hippocampus, and cerebellum of
mice and their human counterparts.
The anatomical characteristics of each of the many species today employed in biomedical research are very important when selecting the correct animal model(s), especially for conducting translational ...research. In previous papers, these features have been considered for fish (D'Angelo et al., 2016), the most common laboratory rodents, rabbits, and pigs (Lossi et al. 2016). I here follow this line of discussion by dealing with the importance of proper knowledge of ferrets, goats, sheep, and horses' main anatomical features in translational research.
Phosphorylation of the histone H2AX (γH2AX form) is an early response to DNA damage and a marker of aging and disease in several cells and tissues outside the nervous system. Little is known about in ...vivo phosphorylation of H2AX in neurons, although it was suggested that γH2AX is an early marker of neuronal endangerment thus opening the possibility to target it as a neuroprotective strategy. After experimental labeling of DNA-synthesizing cells with 5-bromo-2-deoxyuridine (BrdU), we studied the brain occurrence of γH2AX in developing, postnatal, adult and senescent (2 years) mice by light and electron microscopic immunocytochemistry and Western blotting. Focal and/or diffuse γH2AX immunostaining appears in interkinetic nuclei, mitotic chromosomes, and apoptotic nuclei. Immunoreactivity is mainly associated with neurogenetic areas, i.e., the subventricular zone (SVZ) of telencephalon, the cerebellar cortex, and, albeit to a much lesser extent, the subgranular zone of the hippocampal dentate gyrus. In addition, γH2AX is highly expressed in the adult and senescent cerebral cortex, particularly the piriform cortex. Double labeling experiments demonstrate that γH2AX in neurogenetic brain areas is temporally and functionally related to proliferation and apoptosis of neuronal precursors, i.e., the type C transit amplifying cells (SVZ) and the granule cell precursors (cerebellum). Conversely, γH2AX-immunoreactive cortical neurons incorporating the S phase-label BrdU do not express the proliferation marker phosphorylated histone H3, indicating that these postmitotic cells undergo a significant DNA damage response. Our study paves the way for a better comprehension of the role of H2AX phosphorylation in the normal brain, and offers additional data to design novel strategies for the protection of neuronal precursors and mature neurons in central nervous system (CNS) degenerative diseases.
A mutation of the reln gene gives rise to the Reeler mouse (reln (-∕-)) displaying an ataxic phenotype and cerebellar hypoplasia. We have characterized the neurochemistry of postnatal (P0-P60) reln ...(-∕-) mouse cerebella with specific attention to the intervention of cell proliferation and apoptosis in the P0-P25 interval. Homozygous reln (-∕-) mice and age-matched controls were analyzed by immunofluorescence using primary antibodies against NeuN, calbindin, GFAP, vimentin, SMI32, and GAD67. Proliferation and apoptosis were detected after a single intraperitoneal BrdU injection and by the TUNEL assay with anti-digoxigenin rhodamine-conjugated antibodies. Quantitative analysis with descriptive and predictive statistics was used to calculate cell densities (number/mm(2)) after fluorescent nuclear stain (TCD, total cell density), labeling with BrdU (PrCD, proliferating cell density), or TUNEL (ApoCD, apoptotic cell density). By this approach we first have shown that the temporal pattern of expression of neuronal/glial markers in postnatal cerebellum is not affected by the Reeler mutation. Then, we have demonstrated that the hypoplasia in the Reeler mouse cerebellum is consequent to reduction of cortical size and cellularity (TCD), and that TCD is, in turn, linked to quantitative differences in the extent of cell proliferation and apoptosis, as well as derangements in their temporal trends during postnatal maturation. Finally, we have calculated that PrCD is the most important predictive factor to determine TCD in the cerebellar cortex of the mutants. These results support the notion that, beside the well-known consequences onto the migration of the cerebellar neurons, the lack of Reelin results in a measurable deficit in neural proliferation.
Ex vivo spinal cord slice cultures (SCSC) allow study of spinal cord circuitry, maintaining stimuli responses comparable to live animals. Previously, we have shown that mesenchymal stem/stromal cell ...(MSC) transplantation in vivo reduced inflammation and increased nerve regeneration but MSC survival was short-lived, highlighting that beneficial action may derive from the secretome. Previous in vitro studies of MSC conditioned medium (CM) have also shown increased neuronal growth. In this study, murine SCSC were cultured in canine MSC CM (harvested from the adipose tissue of excised inguinal fat) and cell phenotypes analysed via immunohistochemistry and confocal microscopy. SCSC in MSC CM displayed enhanced viability after propidium iodide staining. GFAP immunoreactivity was significantly increased in SCSC in MSC CM compared to controls, but with no change in proteoglycan (NG2) immunoreactivity. In contrast, culture in MSC CM significantly decreased the prevalence of βIII-tubulin immunoreactive neurites, whilst Ca2+ transients per cell were significantly increased. These ex vivo results contradict previous in vitro and in vivo reports of how MSC and their secretome may affect the microenvironment of the spinal cord after injury and highlight the importance of a careful comparison of the different experimental conditions used to assess the potential of cell therapies for the treatment of spinal cord injury.
•Treatment of spinal slices with conditioned medium caused cell phenotypic changes.•Resident astrocytes become hypertrophic, yet neuronal axonal outgrowth reduced.•Signalling cells reduced in number but increased their signalling activity.•Highlights importance of simulation systems and systemic factors in CNS models.
Brain‐derived neurotrophic factor (BDNF) exerts its trophic effects by acting on the high‐affinity specific receptor trkB. BDNF also modulates synaptic transmission in several areas of the CNS, ...including the spinal cord dorsal horn, where it acts as a pain modulator by yet incompletely understood mechanisms. Spinal neurons are the main source of trkB in lamina II (substantia gelatinosa). Expression of this receptor in dorsal root ganglion (DRG) cells has been a matter of debate, whereas a subpopulation of DRG neurons bears trkA receptors and contains BDNF. By the use of two different trkB antibodies we observed that 7.7% and 10.8% of DRG neurons co‐expressed BDNF + trkB but not trkA, respectively, in rat and mouse. Ultrastructurally, full‐length trkB (fl‐trkB) receptors were present at somato‐dendritic membranes of lamina II neurons (rat: 66.8%; mouse: 73.8%) and at axon terminals (rat: 33.2%; mouse: 26.2%). In both species, about 90% of these terminals were identified as primary afferent fibres (PAFs) considering their morphology and/or neuropeptide content. All fl‐trkB‐immunopositive C boutons in type Ib glomeruli were immunoreactive for BDNF and, at individual glomeruli and axo‐dendritic synapses, fl‐trkB receptors were located in a mutually exclusive fashion at pre‐ or postsynaptic membranes. Thus, only a small fraction of fl‐trkB‐immunoreactive dendrites were postsynaptic to BDNF‐immunopositive PAFs. This is the first ultrastructural description of fl‐trkB localization at synapses between first‐ and second‐order sensory neurons in lamina II, and suggests that BDNF may be released by fl‐trkB‐immunopositive PAFs to modulate nociceptive input in this lamina of dorsal horn.
Background: Reelin has fundamental functions in the developing and mature brain. Its absence gives rise to the Reeler phenotype in mice, the first described cerebellar mutation. In homozygous mutants ...missing the Reelin gene (
reln
-/-), neurons are incapable of correctly positioning themselves in layered brain areas such as the cerebral and cerebellar cortices. We here demonstrate that by employing
ex vivo cultured cerebellar slices one can reduce the number of animals and use a non-recovery procedure to analyze the effects of Reelin on the migration of Purkinje neurons (PNs).
Methods: We generated mouse hybrids (L7-GFP
relnF1/) with green fluorescent protein (GFP)-tagged PNs, directly visible under fluorescence microscopy. We then cultured the slices obtained from mice with different
reln genotypes and demonstrated that when the slices from
reln
-/-
mutants were co-cultured with those from reln
+/- mice, the Reelin produced by the latter induced migration of the PNs to partially rescue the normal layered cortical histology. We have confirmed this observation with Voronoi tessellation to analyze PN dispersion.
Results: In images of the co-cultured slices from
reln
-/-
mice, Voronoi polygons were larger than in single-cultured slices of the same genetic background but smaller than those generated from slices of
reln
+/-
animals. The mean roundness factor, area disorder, and roundness factor homogeneity were different when slices from
reln
-/-
mice were cultivated singularly or co-cultivated, supporting mathematically the transition from the clustered organization of the PNs in the absence of Reelin to a layered structure when the protein is supplied
ex vivo.
Conclusions: Neurobiologists are the primary target users of this 3Rs approach. They should adopt it for the possibility to study and manipulate
ex vivo the activity of a brain-secreted or genetically engineered protein (scientific perspective), the potential reduction (up to 20%) of the animals used, and the total avoidance of severe surgery (3Rs perspective).
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