Assessment of cone beam computed tomography techniques for imaging lung damage in mice in vivo

Lung damage is a common side effect of chemotherapeutic drugs such as bleomycin. This study used a bleomycin mouse model which simulates the lung damage observed in humans. Noninvasive, in vivo cone-beam computed tomography (CBCT) was used to visualize and quantify fibrotic and inflammatory damage over the entire lung volume of mice. Bleomycin was used to induce pulmonary damage in vivo and the results from two CBCT systems, a micro-CT and flat panel CT (fpCT), were compared to histologic measurements, the standard method of murine lung damage quantification. Twenty C57BL/6 mice were given either 3 U/kg of bleomycin or saline intratracheally. The mice were scanned at baseline, before the administration of bleomycin, and then 10, 14, and 21 days afterward. At each time point, a subset of mice was sacrificed for histologic analysis. The resulting CT images were used to assess lung volume. Percent lung damage (PLD) was calculated for each mouse on both the fpCT (PLDfpcT) and the micro-CT (PLDμCT). Histologic PLD (PLDH) was calculated for each histologic section at each time point (day 10, n = 4; day 14, n = 4; day 21, n = 5; control group, n = 5). A linear regression was applied to the PLDfpCT vs. PLDH, PLDμCT vs. PLDH and PLDfpCT vs. PLDμCT distributions. This study did not demonstrate strong correlations between PLDCT and PLDH. The coefficient of determination, R, was 0.68 for PLDμCT vs. PLDH and 0.75 for the PLD fpCT vs. PLDH. The experimental issues identified from this study were: (1) inconsistent inflation of the lungs from scan to scan, (2) variable distribution of damage (one histologic section not representative of overall lung damage), (3) control mice not scanned with each group of bleomycin mice, (4) two CT systems caused long anesthesia time for the mice, and (5) respiratory gating did not hold the volume of lung constant throughout the scan. Addressing these issues might allow for further improvement of the correlation between PLDCT and PLDH. ^

Regulation of T cell function by complement C5A in mice during mycobacterial infection

Tuberculosis is the leading cause of death in the world due to a single infectious agent, making it critical to investigate all aspects of the immune response mounted against the causative agent, Mycobacterium tuberculosis , in order to better treat and prevent disease. Previous observations show a disparity in the ability to control mycobacterial growth between mouse strains sufficient in C5, such as C57BL/6 and B10.D2/nSnJ, and those naturally deficient in C5, such as A/J and B10.D2/nSnJ, with C5 deficient mice being more susceptible. It has been shown that during M. tuberculosis infection, C5 deficient macrophages have a defect in production of interleukin (IL)-12, a cytokine involved in the cyclical activation between infected macrophages and effector T cells. T cells stimulated by IL-12 produce interferon (IFN)-γ, the signature cytokine of T helper type 1 (Th1) cells. It is known that a cell-mediated Th1 response is crucial for control of M. tuberculosis in the lungs of humans and mice. This study demonstrates that murine T cells express detectable levels of CD88, a receptor for C5a (C5aR), following antigen presentation by macrophages infected with mycobacteria. T cells from C5 deficient mice infected with M. tuberculosis were found to secrete less IFN-γ and had a reduced Th1 phenotype associated with fewer cells expressing the transcription factor, T-box expressed in T cells (T-bet). The altered Th1 phenotype in M. tuberculosis infected C5 deficient mice coincided with a rise in IL-4 and IL-10 secretion from Th2 cells and inducible regulatory T cells, respectively. It was found that the ineffective T cell response to mycobacteria in C5 deficient mice was due indirectly to a lack of C5a via poor priming by infected macrophages and possibly by a direct interaction between T cells and C5a peptide. Therefore, these studies show a link between the cells of the innate and adaptive arms of the immune system, macrophages and T cells respectively, that was mediated by C5a using a mouse model of M. tuberculosis infection. ^

The diagnostic value of a 12-lead electrocardiogram in patients with cardiac tamponade due to a malignant pericardial effusion

Background. Cardiac tamponade can occur when a large amount of fluid, gas, singly or in combination, accumulating within the pericardium, compresses the heart causing circulatory compromise. Although previous investigators have found the 12-lead ECG to have a poor predictive value in diagnosing cardiac tamponade, very few studies have evaluated it as a follow up tool for ruling in or ruling out tamponade in patients with previously diagnosed malignant pericardial effusions. ^ Methods. 127 patients with malignant pericardial effusions at the MD Anderson Cancer Center were included in this retrospective study. While 83 of these patients had a cardiac tamponade diagnosed by echocardiographic criteria (Gold standard), 44 did not. We computed the sensitivity (Se), specificity (Sp), positive (PPV) and negative predictive values (NPV) for individual and combinations of ECG abnormalities. Individual ECG abnormalities were also entered singly into a univariate logistic regression model to predict tamponade. ^ Results. For patients with effusions of all sizes, electrical alternans had a Se, Sp, PPV and NPV of 22.61%, 97.61%, 95% and 39.25% respectively. These parameters for low voltage complexes were 55.95%, 74.44%, 81.03%, 46.37% respectively. The presence of all three ECG abnormalities had a Se = 8.33%, Sp = 100%, PPV = 100% and NPV = 35.83% while the presence of at least one of the three ECG abnormalities had a Se = 89.28%, Sp = 46.51%, PPV = 76.53%, NPV = 68.96%. For patients with effusions of all sizes electrical alternans had an OR of 12.28 (1.58–95.17, p = 0.016), while the presence of at least one ECG abnormality had an OR of 7.25 (2.9–18.1, p = 0.000) in predicting tamponade. ^ Conclusions. Although individual ECG abnormalities had low sensitivities, specificities, NPVs and PPVs with the exception of electrical alternans, the presence of at least one of the three ECG abnormalities had a high sensitivity in diagnosing cardiac tamponade. This could point to its potential use as a screening test with a correspondingly high NPV to rule out a diagnosis of tamponade in patients with malignant pericardial effusions. This could save expensive echocardiographic assessments in patients with previously diagnosed pericardial effusions. ^

STUDIES ON CHOLINERGIC FUNCTION AND MEMORY IN MICE (CHOLINOTOXINS, WORKING, REFERENCE MEMORY, RADIAL ARM MAZE)

Considerable evidence suggests that central cholinergic neurons participate in either acquisition, storage or retrieval of information. Experiments were designed to evaluate information processing in mice following either reversible or irreversible impairment in central cholinergic activity. The cholinergic receptor antagonists, atropine and methylatropine were used to reversibly inhibit cholinergic transmission. Irreversible impairment in central cholinergic function was achieved by central administration of the cholinergic-specific neurotoxins, N-ethyl-choline aziridinium (ECA) and N-ethyl-acetylcholine aziridinium (EACA).^ ECA and EACA appear to act by irreversible inhibition of high affinity choline uptake (proposed rate-limiting step in acetylcholine synthesis). Intraventricular administration of ECA or EACA produced persistent reduction in hippocampal choline acetyltransferase activity. Other neuronal systems and brain regions showed no evidence of toxicity.^ Mice treated with either ECA or EACA showed behavioral deficits associated with cholinergic dysfunction. Passive avoidance behavior was significantly impaired by cholinotoxin treatment. Radial arm maze performance was also significantly impaired in cholinotoxin-treated animals. Deficits in radial arm maze performance were transient, however, such that rapid and apparent complete behavioral recovery was seen during retention testing. The centrally active cholinergic receptor antagonist atropine also caused significant impairment in radial arm maze behavior, while equivalent doses of methylatropine were without effect.^ The relative effects of cholinotoxin and receptor antagonist treatment on short-term (working) memory and long-term (reference) memory in radial arm maze behavior were examined. Maze rotation studies indicated that there were at least two different response strategies which could result in accurate maze performance. One strategy involved the use of response algorithms and was considered to be a function of reference memory. Another strategy appeared to be primarily dependent on spatial working memory. However, all behavioral paradigms with multiple trails have reference memory requirements (i.e. information useful over all trials). Performance was similarly affected following either cholinotoxin or anticholinergic treatment, regardless of the response strategy utilized. In addition, rates of behavioral recovery following cholinotoxin treatment were similar between response groups. It was concluded that both cholinotoxin and anticholinergic treatment primarily resulted in impaired reference memory processes. ^

AN INVESTIGATION OF THE MODIFYING EFFECTS OF VITAMIN A AND HYPOXIC CELL SENSITIZERS IN RADIATION CARCINOGENESIS IN MICE

The effect of vitamin A (retinyl acetate) and three hypoxic cell sensitizers (metronidazole, misonidazole and desmethylmisonidazole) on lung tumor development in strain A mice exposed to radiation was assessed.^ In experiments involving vitamin A, two groups of mice were fed a low vitamin A diet (< 100 IU/100g diet) while the two other groups were fed a high vitamin A diet (800 IU/100g diet). After two weeks one group maintained on the high vitamin A diet and one group maintained on the low vitamin A diet were given an acute dose of 500 rad of gamma radiation to the thoracic region. The circulating level of plasma vitamin A in all four groups of mice was monitored. A difference in circulating vitamin A in the mice maintained on high and low vitamin A diet became evident by 20 weeks and continued for the duration of the experiment. Mice were killed 18, 26, and 40 weeks post irradiation, their lungs were removed and the number of surface adenomas were counted. There was a significant increase in the number of mice bearing lung tumors and the mean number of lung tumors per mouse in the irradiated group maintained on the high vitamin A diet at 40 weeks post irradiation as compared to the irradiated group maintained on a low vitamin A diet (p < 0.05). Under the conditions of this experiment the development of pulmonary adenomas in irradiated strain A mice appears to relate directly to circulating levels of vitamin A.^ In the other experiment two dose levels of the hypoxic cell sensitizers, 0.2mg/g and 0.6mg/g, were used either alone or in combination with 900 rad of gamma radiation in a fractionated dose schedule of twice a week for three weeks. In the groups of mice which received hypoxic cell sensitizers only, the prevalence and the mean number of lung tumors per mouse were somewhat increased (p < 0.10) in the higher dose group (0.6mg/g) of misonidazole but was not significantly different from the control animals in the other two sensitizer groups. The combination of hypoxic cell sensitizer and radiation did not show any significant enhancement of lung tumor response when compared with the group which received radiation only. The dose of radiation used in this study significantly enhanced lung tumor formation in mice when compared with the control group. Thus, under the experimental exposure conditions used in this investigation, which were very similar to the exposure conditions occurring in clinical treatment, all three hypoxic cell sensitizers did not sensitize the mouse to the carcinogenic effects of gamma radiation.^

Acceleration of the PanIN Development in Mice Expressing Oncogenic K-Ras Due to a High Fat Diet

Obesity is postulated to be one of the major risk factors for pancreatic cancer, and recently it was indicated that an elevated body mass index (BMI correlates strongly with a decrease in patient survival. Despite the evident relationship, the molecular mechanisms involved are unclear. Oncogenic mutation of K-Ras is found early and is universal in pancreatic cancer. Extensive evidence indicates oncogenic K-Ras is not entirely active and it requires a triggering event to surpass the activity of Ras beyond the threshold necessary for a Ras-inflammation feed-forward loop. We hypothesize that high fat intake induces a persistent low level inflammatory response triggering increased K-Ras activity and that Cox-2 is essential for this inflammatory reaction. To determine this, LSL-K-Ras mice were crossed with Ela-CreER (Acinar-specific) or Pdx-1-Cre (Pancreas-specific) to “knock-in” oncogenic K-Ras. Additionally, these animals were crossed with Cox-2 conditional knockout mice to access the importance of Cox-2 in the inflammatory loop present. The mice were fed isocaloric diets containing 60% energy or 10% energy from fat. We found that a high fat diet increased K-Ras activity, PanIN formation, and fibrotic stroma significantly compared to a control diet. Genetic deletion of Cox-2 prevented high fat diet induced fibrosis and PanIN formation in oncogenic K-Ras expressing mice. Additionally, long term consumption of high fat diet, increased the progression of PanIN lesions leading to invasive cancer and decreased overall survival rate. These findings indicate that a high fat diet can stimulate the activation of oncogenic K-Ras and initiate an inflammatory feed forward loop requiring Cox-2 leading to inflammation, fibrosis, and PanINs. This mechanism could explain the relationship between a high fat diet and elevated risk for pancreatic cancer.

Altered tumorigenic phenotype in mice with a hypomorphic p53 mutant allele

Mutations in the p53 tumor suppressor gene are found in over 50% of human tumors and in the germline of Li-Fraumeni syndrome families. About 80% of these mutations are missense in nature. In order to study how p53 missense mutations affect tumorigenesis in vivo, we focused on the murine p53 arg-to-his mutation at amino acid 172, which corresponds to the human hot spot mutation at amino acid 175. The double replacement procedure was employed to introduce the p53 R172H mutation into the p53 locus of ES cells and mice were generated. An additional 1bp deletion in the intron 2 splice acceptor site was detected in the same allele in mice. We named this allele p53R172HΔg. This allele makes a small amount of full length p53 mutant protein. ^ Spontaneous tumor formation and survival were studied in these mice. Mice heterozygous for the p53R172HΔg allele showed 50% survival at 17 months of age, similar to the p53+/− mice. Moreover, the p53R172HΔg/+ mice showed a distinct tumor spectrum: 55% sarcomas, including osteosarcoms, fibrosarcomas and angiosarcomas; 27% carcinomas, including lung adenocarcinomas, squamous cell carcinomas, hepatocellular carcinomas and islet cell carcinomas; and 18% lymphomas. Compared to the p53+/− mice, there was a clear increase in the frequency of carcinoma development and a decrease in lymphoma incidence. Among the sarcomas that developed, fibrosarcomas in the skin were also more frequently observed. More importantly, osteosarcomas and carinomas that developed in the p53R172HΔg/+ mice metastasized at very high frequency (64% and 67%, respectively) compared with less than 10% in the p53+/− mice. The metastatic lesions were usually found in lung and liver, and less frequently in other tissues. The altered tumor spectrum in the mice and increased metastatic potential of the tumors suggested that the p53R172H mutation represents a gain-of-function. ^ Mouse embryonic fibroblasts (MEFs) from the mice homozygous and heterozygous for the p53R172HΔg allele were studied for growth characteristics, immortalization potential and genomic instability. All of the p53R172HΔg /+ MEF lines are immortalized under a 3T3 protocol while under the same protocol p53+/− MEFs are not immortalized. Karyotype analysis showed a persistent appearance of chromosome end-to-end fusion in the MEFs both homozygous and heterozygous for the p53R172HΔg allele. These observations suggest that increased genomic instability in the cells may cause the altered tumor phenotypes. ^

Pathogenetic sequence for aneurysm revealed in mice underexpressing fibrillin-1

Dissecting aortic aneurysm is the hallmark of Marfan syndrome (MFS) and the result of mutations in fibrillin-1, the major constituent of elastin-associated extracellular microfibrils. It is yet to be established whether dysfunction of fibrillin-1 perturbs the ability of the elastic vessel wall to sustain hemodynamic stress by disrupting microfibrillar assembly, by impairing the homeostasis of established elastic fibers, or by a combination of both mechanisms. The pathogenic sequence responsible for the mechanical collapse of the elastic lamellae in the aortic wall is also unknown. Targeted mutation of the mouse fibrillin-1 gene has recently suggested that deficiency of fibrillin-1 reduces tissue homeostasis rather than elastic fiber formation. Here we describe another gene-targeting mutation, mgR, which shows that underexpression of fibrillin-1 similarly leads to MFS-like manifestations. Histopathological analysis of mgR/mgR specimens implicates medial calcification, the inflammatory–fibroproliferative response, and inflammation-mediated elastolysis in the natural history of dissecting aneurysm. More generally, the phenotypic severity associated with various combinations of normal and mutant fibrillin-1 alleles suggests a threshold phenomenon for the functional collapse of the vessel wall that is based on the level and the integrity of microfibrils.

Impaired fetal T cell development and perinatal lethality in mice lacking the cAMP response element binding protein

CREB, the cAMP response element binding protein, is a key transcriptional regulator of a large number of genes containing a CRE consensus sequence in their upstream regulatory regions. Mice with a hypomorphic allele of CREB that leads to a loss of the CREBα and Δ isoforms and to an overexpression of the CREBβ isoform are viable. Herein we report the generation of CREB null mice, which have all functional isoforms (CREBα, β, and Δ) inactivated. In contrast to the CREBαΔ mice, CREB null mice are smaller than their littermates and die immediately after birth from respiratory distress. In brain, a strong reduction in the corpus callosum and the anterior commissures is observed. Furthermore, CREB null mice have an impaired fetal T cell development of the αβ lineage, which is not affected in CREBαΔ mice on embryonic day 18.5. Overall thymic cellularity in CREB null mice is severely reduced affecting all developmental stages of the αβ T cell lineage. In contrast γδ T cell differentiation is normal in CREB mutant mice.

Malignant transformation of early lymphoid progenitors in mice expressing an activated Blk tyrosine kinase

The intracellular signals governing cellular proliferation and developmental progression during lymphocyte development are incompletely understood. The tyrosine kinase Blk is expressed preferentially in the B lineage, but its function in B cell development has been largely unexplored. We have generated transgenic mice expressing constitutively active Blk [Blk(Y495F)] in the B and T lymphoid compartments. Expression of Blk(Y495F) in the B lineage at levels similar to that of endogenous Blk induced B lymphoid tumors of limited clonality, whose phenotypes are characteristic of B cell progenitors at the proB/preB-I to preB-II transition. Expression of constitutively active Blk in the T lineage resulted in the appearance of clonal, thymic lymphomas composed of intermediate single positive cells. Taken together, these results indicate that specific B and T cell progenitor subsets are preferentially susceptible to transformation by Blk(Y495F) and suggest a role for Blk in the control of proliferation during B cell development.

Resistance to endotoxic shock and reduced neutrophil migration in mice deficient for the Src-family kinases Hck and Fgr

Signal transduction through the leukocyte integrins is required for the processes of firm adhesion, activation, and chemotaxis of neutrophils during inflammatory reactions. Neutrophils isolated from knockout mice that are deficient in the expression of p59/61hck (Hck) and p58c-fgr (Fgr), members of the Src-family of protein tyrosine kinases, have been shown to be defective in adhesion mediated activation. Cells from these animals have impaired induction of respiratory burst and granule secretion following plating on surfaces that crosslink β2 and β3 integrins. To determine if the defective function of hck−/−fgr−/− neutrophils observed in vitro also results in impaired inflammatory responses in vivo, we examined responses induced by lipopolysaccharide (LPS) injection in these animals. The hck−/−fgr−/− mice showed marked resistance to the lethal effects of high-dose LPS injection despite the fact that high levels of serum tumor necrosis factor α and interleukin 1α were detected. Serum chemistry analysis revealed a marked reduction in liver and renal damage in mutant mice treated with LPS, whereas blood counts showed a marked neutrophilia that was not seen in wild-type animals. Direct examination of liver sections from mutant mice revealed reduced neutrophil migration into the tissue. These data demonstrate that defective integrin signaling in neutrophils, caused by loss of Hck and Fgr tyrosine kinase activity, results in impaired inflammation-dependent tissue injury in vivo.

Prothrombin deficiency results in embryonic and neonatal lethality in mice

The conversion of prothrombin (FII) to the serine protease, thrombin (FIIa), is a key step in the coagulation cascade because FIIa triggers platelet activation, converts fibrinogen to fibrin, and activates regulatory pathways that both promote and ultimately suppress coagulation. However, several observations suggest that FII may serve a broader physiological role than simply stemming blood loss, including the identification of multiple G protein-coupled, thrombin-activated receptors, and the well-documented mitogenic activity of FIIa in in vitro test systems. To explore in greater detail the physiological roles of FII in vivo, FII-deficient (FII−/−) mice were generated. Inactivation of the FII gene leads to partial embryonic lethality with more than one-half of the FII−/− embryos dying between embryonic days 9.5 and 11.5. Bleeding into the yolk sac cavity and varying degrees of tissue necrosis were observed in many FII−/− embryos within this gestational time frame. However, at least one-quarter of the FII−/− mice survived to term, but ultimately they, too, developed fatal hemorrhagic events and died within a few days of birth. This study directly demonstrates that FII is important in maintaining vascular integrity during development as well as postnatal life.

Incomplete embryonic lethality and fatal neonatal hemorrhage caused by prothrombin deficiency in mice

Deficiency of blood coagulation factor V or tissue factor causes the death of mouse embryos by 10.5 days of gestation, suggesting that part of the blood coagulation system is necessary for development. This function is proposed to require either generation of the serine protease thrombin and cell signaling through protease-activated receptors or an activity of tissue factor that is distinct from blood clotting. We find that murine deficiency of prothrombin clotting factor 2 (Cf2) was associated with the death of approximately 50% of Cf2−/− embryos by embryonic day 10.5 (E10.5), and surviving embryos had characteristic defects in yolk sac vasculature. Most of the remaining Cf2−/− embryos died by E15.5, but those surviving to E18.5 appeared normal. The rare Cf2−/− neonates died of hemorrhage on the first postnatal day. These studies suggest that a part of the blood coagulation system is adapted to perform a developmental function. Other mouse models show that the absence of platelets or of fibrinogen does not cause fetal wastage. Therefore, the role of thrombin in development may be independent of its effects on blood coagulation and instead may involve signal transduction on cells other than platelets.

Delayed embryonic lethality in mice lacking protein phosphatase 2A catalytic subunit Cα

Protein phosphatase 2A (PP2A) is a multimeric enzyme, containing a catalytic subunit complexed with two regulatory subunits. The catalytic subunit PP2A C is encoded by two distinct and unlinked genes, termed Cα and Cβ. The specific function of these two catalytic subunits is unknown. To address the possible redundancy between PP2A and related phosphatases as well as between Cα and Cβ, the Cα subunit gene was deleted by homologous recombination. Homozygous null mutant mice are embryonically lethal, demonstrating that the Cα subunit gene is an essential gene. As PP2A exerts a range of cellular functions including cell cycle regulation and cell fate determination, we were surprised to find that these embryos develop normally until postimplantation, around embryonic day 5.5/6.0. While no Cα protein is expressed, we find comparable expression levels of PP2A C at a time when the embryo is degenerating. Despite a 97% amino acid identity, Cβ cannot completely compensate for the absence of Cα. Degenerated embryos can be recovered even at embryonic day 13.5, indicating that although embryonic tissue is still capable of proliferating, normal differentiation is significantly impaired. While the primary germ layers ectoderm and endoderm are formed, mesoderm is not formed in degenerating embryos.

Resistance to DNA fragmentation and chromatin condensation in mice lacking the DNA fragmentation factor 45

The DNA fragmentation factor 45 (DFF45) is a subunit of a heterodimeric nuclease complex critical for the induction of DNA fragmentation in vitro. To understand the in vivo role of DFF45 in programmed cell death, we generated DFF45 mutant mice. DNA fragmentation activity is completely abolished in cell extracts from DFF45 mutant tissues. In response to apoptotic stimuli, splenocytes, thymocytes, and granulocytes from DFF45 mutant mice are resistant to DNA fragmentation, and splenocytes and thymocytes are also resistant to chromatin condensation. Nevertheless, development of the immune system in the DFF45 mutant mice is normal. These results demonstrate that DFF45 is critical for the induction of DNA fragmentation and chromatin condensation in vivo, but is not required for normal immune system development.