Mice as laboratory animals

Troponin I is a contractile protein and plays an important role in cardiac function.
We have generated cTnl knockout and cTnI(R192H) transgenic mouse models. All of
cTnl knockout homozygous mice die at 17-18 days after birth. Some of cTnI(R192H)
transgenic mice die at early life stages, some mice develop heart failure at late stages.
High-resolution ultrasound imaging and Doppler echocardiography have been used to
evaluate cardiac function on cTnl deficient mice and cTnl(R192H) transgenic mice.
cTnI mice have damaged relaxation with gradually decreased E/A ratio(E/A<1). FS
and cardiac output dramatically decrease on 17-day-o1d cTnI mice indicating severe
cardiac dysfunction. We find that the damaged heart function is correspondent with the
Tnl expression level decline. 6-8 weeks transgenic mice have shown that the dimension
of left and right atria increase. In 15-month-old transgenic mice, the E/A ratio shows a
pseudonormal pattern indicating a diastolic dysfunction. This study demonstrate that
damaged heart function is tightly associated with Tnl levels in the heart.

It is of interest to understand how new neurons incorporate themselves into the
existing circuitry of certain neuronal populations. One such population of neurons is that
which are born in the subventricular zone (SVZ) and migrate to the olfactory bulb where
they differentiate into granule cells. Another area of interest is the role of brain-derived
neurotrophic factor (BDNF) on the survival and overall health of these neurons. This
study aimed to test whether or not BDNF is a survival factor for adult-born granule cells.
Here were utilized a transgenic mouse model over-expressing BDNF under the α-
calcium/calmodulin-dependent protein kinase II (CAMKIIα) promoter, and tested its
effect on olfactory granule cells under sensory deprived conditions. Results from this
experiment indicated that there was no significant difference in cell death or cell survival when comparing transgenic and wild type animals. We concluded that BDNF is not a
survival factor for adult-born granule cells.

Metastasis is the primary cause of mortality in women with breast cancer. Recently, elevated serum levels of a glycoprotein known as chitinase-3 likeprotein- 1 (CHI3L1) has been correlated with poor prognosis and shorter survival of patients with cancer and inflammatory diseases. The biological and physiological functions of CHI3L1 in tumor progression have not yet been elucidated. In this document, we describe the role of CHI3L1 in tumor growth and metastasis and its relationship with inflammation.
Using well-established models of breast cancer, we show that CHI3L1 is increased in the serum of tumor bearing mice. We found that CHI3L1 levels are increased at both the “pre-metastatic” and “metastatic stage” and that tumor cells, splenic, alveolar and interstitial macrophages; and myeloid derived population produce CHI3L1. Furthermore, we demonstrated that CHI3L1 has an inhibitory role on the expression of interferon-gamma (IFN γ) by T cells, while enhancing the production of pro-inflammatory mediators by macrophages such as Cchemokine ligand 2 (CCL2/MCP-1), Chemokine CX motif ligand 2 (CXCL2/IL-8) and matrix metalloproteinase-9 (MMP-9), all of which promote tumor growth and metastasis. We demonstrated that in vivo treatment of tumor-bearing mice with chitin microparticles, a TH1 adjuvant and a substrate for CHI3L1, promoted immune effector functions with increased production of IFN-γ but decreased CCL2/MCP-1, CXCL2/IL-8 and MMP-9 expression by splenic and pulmonary macrophages. Significantly, in vivo administration of chitin microparticles decreased tumor growth and pulmonary metastasis in mammary tumor bearing mice. These results suggest that CHI3L1 may play a role in tumor progression. Inflammation plays a pivotal role during tumor progression and metastasis by promoting the production of pro-inflammatory molecules such as CHI3L1. However, little is known about how CHI3L1 expression can affect secondary sites to enhance metastasis. In these studies, we demonstrated that CHI3L1 alters the cellular composition and inflammatory mediators that aid in the establishment of a metastatic niche for the support of infiltrating tumor cells leading to accelerated tumor progression. Since previous studies showed that CHI3L1 modulates inflammation, we determined the role of CHI3L1 in the context of pre-existing inflammation and metastasis. We found that CHI3L1 deficient mice with preexisting inflammation had decreased pro-inflammatory mediators, and significant reduction in tumor volume and metastasis compared to wild type controls. Preexisting inflammation and CHI3L1 may be driving the establishment of a premetastatic milieu in the lungs and aiding in the establishment of metastasis. Understanding the role of CHI3L1 in inflammation during tumor progression could result in the design of targeted therapies for breast cancer patients.

Small conductance Ca2+-activated K+ (SK) channels have been shown to alter the encoding of spatial and non-spatial memory in the hippocampus by shaping glutamatergic postsynaptic potentials and modulating NMDA receptor-dependent synaptic plasticity. When activated, dendritic SK channels reduce hippocampal neuronal excitability and LTP. Similar SK channel properties have been demonstrated in lateral amygdala (LA) pyramidal neurons. Additionally, induction of synaptic plasticity and beta-adrenoreceptor activation in LA pyramidal neurons causes PKA-mediated internalization of SK channels from the postsynaptic density. Chronic activation of the amygdala through repetitive stressful stimuli can lead to excitatory synaptic strengthening that may create permanent hyper-excitability in its circuitry. This mechanism may contribute to a number of mood and anxiety disorders. The selective influence of SK channels in the LA on anxiety and fear conditioning are not known. The thesis project outlined herein examined whether SK channel blockade by bee venom peptide, apamin, during a repetitive acute fear conditioning paradigm was sufficient to alter fear memory encoding and the resulting behavioral outcome. Following the final fear memory test session, mice were tested in the open field immediately after the second fear conditioning test session. The findings indicate that intracranial LA microinfusions of apamin did not affect memory encoding or subsequent anxiety.

Transcription and translation of proteins are required for the consolidation of episodic memory. Arc, an effector immediate early gene, has been linked to synaptic plasticity following learning and memory. It is well established that the rodent hippocampus is essential for processing spatial memory, but its role in processing object memory is a point of contention. Using immunohistochemical techniques, hippocampal sections were stained for arc proteins in the CA1 region of the dorsal hippocampus in mice following two variations of the novel object recognition (NOR) task. Results suggest mice that acquired strong object memory showed significant hippocampal activation. In mice that acquired weak object memory, hippocampal activation was not significantly different from controls. Arc expression was also examined in other hippocampal sub-regions, as well as in the perirhinal cortex. These results suggest that the mice must acquire a threshold amount of object information before the hippocampal CA1 region is engaged.

Carbohydrate recognition is one of the most sophisticated recognition processes in biological
systems, mediating many important aspects of cell-cell recognition, such as inflammation, cell
differentiation, and metastasis. Consequently, lectin-glycan interactions have been intensively
studied in order to mimic their actions for potential bioanalytical and biomedical applications.
Galectins, a class of ß-galactoside-specific animal lectins, have been strongly implicated in
inflammation and cancer. Galectin-3 is involved in carbohydrate-mediated metastatic cell
heterotypic and homotypic adhesion via interaction with Thomsen-Friedenreich (TF) antigen on
cancer-associated MUC1. However, the precise mechanism by which galectin-3 recognizes TF
antigen is poorly understood. Our thermodynamic studies have shown that the presentation of the
carbohydrate ligand by MUC1-based peptide scaffolds can have a major impact on recognition,
and may facilitate the design of more potent and specific galectin-3 inhibitors that can be used as
novel chemical tools in dissecting the precise role of galectin-3 in cancer and inflammatory
diseases. Another lectin, odorranalectin (OL), has been recently identified from Odorrana grahami
skin secretions as the smallest cyclic peptide lectin, has a particular selectivity for L-fucose and
very low toxicity and immunogenicity, rendering OL an excellent candidate for drug delivery to
targeted sites, such as: (1) tumor-associated fucosylated antigens implicated in the pathogenesis
of several cancers, for overcoming the nonspecificity of most anticancer agents; (2) the olfactory epithelium of nasal mucosa for enhanced delivery of peptide-based drugs to the brain.

Restrictive cardiomyopathy (RCM) is represented in part by left ventricular stiffness
and diastolic dysfunction. Missense mutations of the cardiac troponin I (cTnI) gene cause
idiopathic RCM. These mutations are located in the C-terminus of cTnI and affect cardiac
relaxation. Transgenic mouse models presenting the pathology observed in clinical
patients with RCM have been generated previously and express the mutant cTnI in their
hearts. RCM-linked mutations increase cardiac myofilament Ca2+ sensitivity and promote
diastolic dysfunction in the heart. Previous studies using double transgenic mice
(cTnI/R193H/ND) showed that ventricular relaxation is enhanced in the cTnI/R193H
transgenic mice. In this study, another double transgenic mouse model,
(cTnI/R193H/ND/KO), provides an avenue to investigate its rescuing effects on RCMlinked
mutations in the cTnI /R193H/KO mouse. Use of molecular biological techniques,
transgenic animal developments and murine echocardiography in this study has
culminated into a greater understanding of RCM and diastolic dysfunction.

The murine strains BALB and C57 differ in behavioral sensitivity to opioid alkaloids, beta-endorphin, and met-/leu-enkephalin. Possible differences in levels of endogenous "opioid peptides" have not been evaluated in the BALB and C57 murine genotypes by the use of immunohistochemical procedures. Antisera to synthetic ovine beta-endorphin was used to examine neuropil and somatic immunoreactivity in various diencephalic and telencephalic areas within three strains of inbred mice, BALB/c, C57BL and the heterogenous strain (CF1). Results indicate that quantitative differences exist between these strains in the number of immunoreactive cells present in the arcuate nucleus of the hypothalamus and in neuropil straining. These observations indicate that behavioral differences in opioid responsivity may relate to differences in either the number of parent somata in the arcuate nucleus and/or the organization and/or trajectory of efferent projections arising from arcuate nucleus neurons.

Olfactory sensory neurons (OSN) expressing the same odor receptor (OR) project their axons to topographically fixed glomeruli in the olfactory bulb (OB). This topographic map results from axon guidance mechanisms determined by ORs, glia and molecular guidance cues. The present study examined the organization of mature OSNs expressing the P2 OR in adult mice after ablation of bulb neurons with N-methyl-D-aspartate (NMDA). Rapid neuronal degeneration was followed by progressive laminar disorganization of the OB and glomerular shrinkage. P2 axon targeting and convergence was maintained within degenerating glomeruli for up to 2 weeks. After that time, fewer P2 axons were observed in the lesioned OB with fewer P2 neurons in the olfactory epithelium (OE). By 3 weeks, the mature OSN population was reduced and the immature population was increased. These results suggest that bulbar synaptic contacts do not maintain sensory axon convergence in the adult, but regulate neuronal survival in the OE.

Troponin I (TnI) plays an important role in cardiac muscle contraction. Two TnI genes (cardiac and slow skeletal TnI) are predominantly expressed in the heart. In cTnI knockout mice, myocardial TnI deficiency results in a diastolic dysfunction and a sudden death in homozygous mutants. In the present studies, energy metabolism has been analyzed in myocardial cells from cTnI null hearts. Our results have demonstrated that damaged relaxation and increased Ca2+-independent force production in cTnI null hearts stimulated myofibril MgATPase activities accompanied by the increase of mitochondria quantity and ATPase activities. In addition, an increase of ssTnI phosphorylation level has been observed in cTnI null hearts. The results indicate that TnI deficiency can cause the disturbance of energy metabolism and some protein overphosphorylation.