Progressive tremor and motor impairment in seizure-prone mutant tremor mice are associated with neurotransmitter dysfunction.

BACKGROUND: The tremor mutant mice present motor impairments comprised of whole-body tremors, ataxia, decreased exploratory behavior, and audiogenic seizures. OBJECTIVES: This study aims to investigate the development of motor dysfunction in this mutant mouse and the relationships with cortical, striatal, and cerebellar levels of GABA, glutamate, glycine, dopamine (DA), serotonin (5-HT), noradrenaline (NOR), and its metabolites. The serum cytokines levels, myelin content, and the astrocytic expression of the glial fibrillary acidic protein (GFAP) investigated the possible influence of inflammation in motor dysfunction. RESULTS: Relative to wild-type (WT) mice, the tremor mice presented: increased tremors and bradykinesia associated with postural instability, decreased range of motion, and difficulty in initiating voluntary movements directly proportional to age; reduced step length for right and left hindlimbs; reduced cortical GABA, glutamate and, aspartate levels, the DOPAC/DA and ratio and increased the NOR levels; in the striatum, the levels of glycine and aspartate were reduced while the HVA levels, the HVA/DA and 5HIAA/5-HT ratios increased; in the cerebellum the glycine, NOR and 5-HIAA levels increased. CONCLUSIONS: We suggest that the motor disturbances resulted mainly from the activation of the indirect striatal inhibitory pathway to the frontal cortex mediated by GABA, glutamate, and aspartate, reducing the dopaminergic activity at the prefrontal cortex, which was associated with the progressive tremor. The reduced striatal and increased cerebellar glycine levels could be partially responsible for the mutant tremor motor disturbances.

Bate palmas mutant mice as a model of Kabuki syndrome: Higher susceptibility to infections and vocalization impairments?

The recessive mutant mouse bate palmas (bapa) arose from N-ethyl-N-nitrosourea mutagenesis. Previous studies of our group revealed some behavioral impairments and a mutation in the lysine (K)-specific methyltransferase 2D (Kmt2d) gene. Because mutations in the KMT2D gene in humans are mainly responsible for Kabuki syndrome, this study was proposed to validate bapa mice as a model of Kabuki syndrome. Besides other symptoms, Kabuki syndrome is characterized by increased susceptibility to infections and speech impairments, usually diagnosed in the early childhood. Thus, juvenile male and female bapa mice were studied in different developmental stages (prepubertal period and puberty). To induce sickness behavior and to study infection susceptibility responses, lipopolysaccharide (LPS) was used. To study oral communication, ultrasonic vocalizations were evaluated. Behavioral (open-field test) and central (astrocytic glial fibrillary acidic protein [GFAP] and tyrosine hydroxylase [TH]) evaluations were also performed. Control and bapa female mice emitted 31-kHz ultrasounds on prepubertal period when exploring a novel environment, a frequency not yet described for mice, being defined as 31-kHz exploratory vocalizations. Males, LPS, and puberty inhibited these vocalizations. Bapa mice presented increased motor/exploratory behaviors on prepubertal period due to increased striatal TH expression, revealing striatal dopaminergic system hyperactivity. Combining open-field behavior and GFAP expression, bapa mice did not develop LPS tolerance, that is, they remained expressing signs of sickness behavior after LPS challenge, being more susceptible to infectious/inflammatory processes. It was concluded that bapa mice is a robust experimental model of Kabuki syndrome.

Pathogenesis of Equid Alphaherpesvirus 1 Infection in the Central Nervous System of Mice.

Equid alphaherpesvirus 1 (EHV-1) causes myeloencephalopathy in horses and occasionally in non-equid species. Although mouse models have been developed to understand EHV-1 pathogenesis, few EHV-1 strains have been identified as highly neurovirulent to mice. The aim of this study was to evaluate the pathogenesis of 2 neurovirulent EHV-1 strains in mice, and to characterize the inflammatory cells and expression of chemokines and the apoptosis marker caspase-3 in the brain of infected mice. C57BL/6J mice were inoculated intranasally with EHV-1 strains A4/72 or A9/92 and evaluated on 1, 2, and 3 days post inoculation (DPI). EHV-1-infected mice showed severe neurological signs at 3 DPI. Ultrastructural analysis revealed numerous viral nucleocapsids and fewer enveloped virions within degenerated and necrotic neurons and in the surrounding neuropil. Histologically, at 3 DPI, there was severe diffuse neuronal degeneration and liquefactive necrosis, prominent microgliosis, and perivascular cuffing composed of CD3+ cells (T cells) and Iba-1+ cells (macrophages), mainly in the olfactory bulb and ventral portions of the brain. In these areas, moderate numbers of neuroglial cells expressed CCL5 and CCL2 chemokines. Numerous neurons, including those in less affected areas, were immunolabeled for cleaved caspase-3. In conclusion, neurovirulent EHV-1 strains induced a fulminant necrotizing lymphohistiocytic meningoencephalitis in mice, with microgliosis and expression of chemokines and caspase-3. This model will be useful for understanding the mechanisms underlying the extensive neuropathology induced by these viral infections.

Equine herpesvirus 1 elicits a strong pro-inflammatory response in the brain of mice.

Equine herpesvirus type 1 (EHV-1) is an emerging pathogen that causes encephalomyelitis in horses and non-equid species. Several aspects of the immune response in the central nervous system (CNS), mainly regarding the role of inflammatory mediators during EHV-1 encephalitis, remain unknown. Moreover, understanding the mechanisms underlying extensive neuropathology induced by viruses would be helpful to establish therapeutic strategies. Therefore, we aimed to evaluate some aspects of the innate immune response during highly neurovirulent EHV-1 infection. C57BL/6 mice infected intranasally with A4/72 and A9/92 EHV-1 strains developed a fulminant neurological disease at 3 days post-inoculation with high viral titres in the brain. These mice developed severe encephalitis with infiltration of monocytes and CD8+ T cells to the brain. The inflammatory infiltrate followed the detection of the chemokines CCL2, CCL3, CCL4, CCL5, CXCL2, CXCL9 and CXCL-10 in the brain. Notably, the levels of CCL3, CCL4, CCL5 and CXCL9 were higher in A4/72-infected mice, which presented higher numbers of inflammatory cells within the CNS. Pro-inflammatory cytokines, such as interleukins (ILs) IL-1α, IL-1ß, IL-6, IL-12ß, and tumour necrosis factor (TNF), were also detected in the CNS, and Toll-like receptor (TLR) TLR2, TLR3 and TLR9 genes were also upregulated within the brain of EHV-1-infected mice. However, no expression of interferon-γ (IFN-γ) and IL-12α, which are important for controlling the replication of other herpesviruses, was detected in EHV-1-infected mice. The results show that the activated innate immune mechanisms could not prevent EHV-1 replication within the CNS, but most likely contributed to the extensive neuropathology. The mouse model of viral encephalitis proposed here will also be useful to study the mechanisms underlying extensive neuropathology.

A Simple and Fast Battery Test for Phenotypic Characterization of Mice.

Despite the great number of test batteries already known to assess the behavior of genetically modified and inbred strains of mice, only a few of them focus on basic neurological parameters. The purpose of the battery test proposed is to settle a specific methodology to characterize the phenotype of neurological disease models in mutant or genetically modified mice. This methodology is simple and efficient in order to analyze several parameters, including general activity, sensory nervous system, sensorimotor system, central nervous system and autonomous nervous system. This can aid the choice of specific additional tests as well as the determination of an interrelationship among phenotypic alterations observed. Although being efficient for a first analysis of a mouse model, interpretation of the results must be carefully made because phenotype manifestation may vary due to many parameters, including mouse strain, environmental and housing condition, animal-experimenter interaction, sample size and tests order. It is important to consider as a critical point if handling procedures are aversive. The results acquired with the analysis of 18 parameters together provide preliminary data to characterize mouse phenotype and helps selecting more specific tests.

Genetic and behavioral characterization of a Kmt2d mouse mutant, a new model for Kabuki Syndrome.

The recessive mutant mice bate palmas (bapa) - claps in Portuguese arose from N-ethyl-N-nitrosourea mutagenesis. A single nucleotide, T > C, change in exon 13, leading to a Thr1289 Ala substitution, was identified in the lysine (K)-specific methyltransferase 2D gene (Kmt2d) located on chromosome 15. Mutations with a loss-of-function in the KMT2D gene on chromosome 12 in humans are responsible for Kabuki syndrome (KS). Phenotypic characterization of the bapa mutant was performed using a behavioral test battery to evaluate the parameters related to general activity, the sensory nervous system, the psychomotor system, and the autonomous nervous system, as well as to measure motor function and spatial memory. Relative to BALB/cJ mice, the bapa mutant showed sensory and psychomotor impairments, such as hypotonia denoted by a surface righting reflex impairment and hindquarter fall, and a reduction in the auricular reflex, suggesting hearing impairment. Additionally, the enhanced general activity showed by the increased rearing and grooming frequency, distance traveled and average speed possibly presupposes the presence of hyperactivity of bapa mice compared with the control group. A slight motor coordination dysfunction was showed in bapa mice, which had a longer crossing time on the balance beam compared with BALB/cJ controls. Male bapa mice also showed spatial gait pattern changes, such as a shorter stride length and shorter step length. In conclusion, the bapa mouse may be a valuable animal model to study the mechanisms involved in psychomotor and behavior impairments, such as hypotonia, fine motor coordination and hyperactivity linked to the Kmt2d mutation.

A Mouse Model for Human Unstable Hemoglobin Santa Ana.

In the present study, we described the phenotype, histologic morphology, and molecular etiology of a mouse model of unstable hemoglobin Santa Ana. Hematologic evaluation of anemic mice (Anem/+) discovered after N-ethyl-N-nitrosourea mutagenesis revealed moderate anemia with intense reticulocytosis and polychromasia, followed by anisocytosis, macrocytosis, hypochromia, and intraerythrocytic inclusion and Heinz bodies. The mice also demonstrated hemoglobinuria, bilirubinemia, and erythrocytic populations with differing resistance to osmotic lysis. Splenomegaly (particularly in older mutant mice) and jaundice were apparent at necropsy. Histopathologic examination revealed dramatically increased hematopoiesis and hemosiderosis in hematopoietic organs and intracellular iron deposition in tubular renal cells. These data are characteristic of a congenital hemolytic regenerative anemia, similar to human anemias due to unstable hemoglobin. Genetic mapping assigned the affected gene to mouse chromosome 7, approximately 50 cM from the Hbb locus. The sequence of the mutant Hbb gene exhibited a T→C transversion at nucleotide 179 in Hbb-b1, leading to the substitution of proline for leucine at amino acid residue 88 and thus homologous to the genetic defect underlying Santa Ana anemia in humans.

Molecular characterization of Brazilian equid herpesvirus type 1 strains based on neuropathogenicity markers.

Partial nucleotide sequences of ORF72 (glycoprotein D, gD), ORF64 (infected cell protein 4, ICP4) and ORF30 (DNA polymerase) genes were compared with corresponding sequences of EHV-1 reference strains to characterize the molecular variability of Brazilian strains. Virus isolation assays were applied to 74 samples including visceral tissue, total blood, cerebrospinal fluid (CSF) and nasal swabs of specimens from a total of 64 animals. Only one CSF sample (Iso07/05 strain) was positive by virus isolation in cell culture. EHV-1 Iso07/05 neurologic strain and two abortion visceral tissues samples (Iso11/06 and Iso33/06) were PCR-positive for ORF33 (glycoprotein B, gB) gene of EHV-1. A sequence analysis of the ORF72, ORF64 and ORF30 genes from three EHV-1 archival strains (A3/97, A4/72, A9/92) and three clinical samples (Iso07/05, Iso11/06 and Iso33/06) suggested that among Brazilian EHV-1 strains, the amplified region of the gD gene sequence is highly conserved. Additionally, the analysis of ICP4 gene showed high nucleotide and amino acid identities when compared with genotype P strains, suggesting that the EHV-1 Brazilian strains belonged to the same group. All the EHV-1 Brazilian strains were classified as non-neuropathogenic variants (N752) based on the ORF30 analysis. These findings indicate a high conservation of the gD-, ICP4- and ORF30-encoding sequences. Different pathotypes of the EHV-1 strain might share identical genes with no specific markers, and tissue tropism is not completely dependent on the gD envelope, immediate-early ICP4 and DNA polymerase proteins.

Molecular characterization of Brazilian equid herpesvirus type 1 strains based on neuropathogenicity markers

Partial nucleotide sequences of ORF72 (glycoprotein D, gD), ORF64 (infected cell protein 4, ICP4) and ORF30 (DNA polymerase) genes were compared with corresponding sequences of EHV-1 reference strains to characterize the molecular variability of Brazilian strains. Virus isolation assays were applied to 74 samples including visceral tissue, total blood, cerebrospinal fluid (CSF) and nasal swabs of specimens from a total of 64 animals. Only one CSF sample (Iso07/05 strain) was positive by virus isolation in cell culture. EHV-1 Iso07/05 neurologic strain and two abortion visceral tissues samples (Iso11/06 and Iso33/06) were PCR-positive for ORF33 (glycoprotein B, gB) gene of EHV-1. A sequence analysis of the ORF72, ORF64 and ORF30 genes from three EHV-1 archival strains (A3/97, A4/72, A9/92) and three clinical samples (Iso07/05, Iso11/06 and Iso33/06) suggested that among Brazilian EHV-1 strains, the amplified region of the gD gene sequence is highly conserved. Additionally, the analysis of ICP4 gene showed high nucleotide and amino acid identities when compared with genotype P strains, suggesting that the EHV-1 Brazilian strains belonged to the same group. All the EHV-1 Brazilian strains were classified as non-neuropathogenic variants (N752) based on the ORF30 analysis. These findings indicate a high conservation of the gD-, ICP4- and ORF30-encoding sequences. Different pathotypes of the EHV-1 strain might share identical genes with no specific markers, and tissue tropism is not completely dependent on the gD envelope, immediate-early ICP4 and DNA polymerase proteins.

Connexin 43 deficiency accelerates skin wound healing and extracellular matrix remodeling in mice.

BACKGROUND: Cellular channels composed of connexin 43 are known to act as key players in the life cycle of the skin and consequently to underlie skin repair. OBJECTIVE: This study was specifically set up to investigate the suite of molecular mechanisms driven by connexin 43-based channels on wound healing. METHODS: To this end, a battery of parameters, including re-epithelialization, neovascularization, collagen deposition and extracellular matrix remodeling, was monitored over time during experimentally induced skin repair in heterozygous connexin 43 knockout mice. RESULTS: It was found that connexin 43 deficiency accelerates re-epithelialization and wound closure, increases proliferation and activation of dermal fibroblasts, and enhances the expression of extracellular matrix remodeling mediators. CONCLUSION: These data substantiate the notion that connexin 43 may represent an interesting therapeutic target in dermal wound healing.

Higher susceptibility of spontaneous and NNK-induced lung neoplasms in connexin 43 deficient CD1 × AJ F1 mice: paradoxical expression of connexin 43 during lung carcinogenesis.

Connexins (Cxs) are proteins that form the communicating gap junctions, and reportedly have a role in carcinogenesis. Here, we evaluated the importance of Connexin43 (Cx43) in spontaneous and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung carcinogenesis. Male wild-type (Cx43(+/+) ) and hemizygote (Cx43(+/-) ) CD1 × AJ F1 mice were injected with NNK or saline. After 60 weeks mice were euthanized; lung nodules were counted, measured, and fixed in formalin or snap frozen. Immunohistochemistry for Cx43 and Beta-catenin (ß-catenin) was performed and Cx43 mRNA expression was evaluated by real-time PCR. Cx43 deletion significantly increased the incidence and number of spontaneous nodules in the CD1 × AJ F1 mice and the number of gross lesions and the aggressiveness of lesions in NNK-treated mice. Cx43 mRNA increased significantly and was correlated with the aggressiveness of tumors, although lesions from Cx43(+/-) mice expressed less Cx43 RNAm than their counterparts. Lung parenchyma presented a Cx43 immunostaining pattern with points or plaques between cells. In hyperplasias and adenomas, Cx43 was found in the membrane and in cytoplasm. Malignant lesions presented increased Cx43 in cytoplasm and a few membrane spots of immunostaining. ß-catenin was weakly expressed in lung parenchyma. Though hyperplasias presented some cells with nuclear ß-catenin, NNK-induced tumors contained a higher number of this staining pattern. Also, no difference in ß-catenin occurred between both genotypes independently of the histological grade. In summary, our results indicate that Cx43 acts as a tumor suppressor gene in early lung tumorigenesis and loses this property in advanced carcinogenesis. Therefore, Cxs are better classified as conditional tumor suppressors.