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PubMed2000

Altered serotonin and dopamine metabolism in the CNS of serotonin 5-HT(1A) or 5-HT(1B) receptor knockout mice.

Measurements of serotonin (5-HT), dopamine (DA), and noradrenaline, and of 5-HT and DA metabolites, were obtained by HPLC from 16 brain regions and the spinal cord of 5-HT(1A) or 5-HT(1B) knockout and wild-type mice of the 129/Sv strain. In 5-HT(1A) knockouts, 5-HT concentrations were unchanged throughout, but levels of 5-HT metabolites were higher than those of the wild type in dorsal/medial raphe nuclei, olfactory bulb, substantia nigra, and locus coeruleus. This was taken as an indication of increased 5-HT turnover, reflecting an augmented basal activity of midbrain raphe neurons and consequent increase in their somatodendritic and axon terminal release of 5-HT. It provided a likely explanation for the increased anxious-like behavior observed in 5-HT(1A) knockout mice. Concomitant increases in DA content and/or DA turnover were interpreted as the result of a disinhibition of DA, whereas increases in noradrenaline concentration in some territories of projection of the locus coeruleus could reflect a diminished activity of its neurons. In 5-HT(1B) knockouts, 5-HT concentrations were lower than those of the wild type in nucleus accumbens, locus coeruleus, spinal cord, and probably also several other territories of 5-HT innervation. A decrease in DA, associated with increased DA turnover, was measured in nucleus accumbens. These changes in 5-HT and DA metabolism were consistent with the increased aggressiveness and the supersensitivity to cocaine reported in 5-HT(1B) knockout mice. Thus, markedly different alterations in CNS monoamine metabolism may contribute to the opposite behavioral phenotypes of these two knockouts.

3,4-Dihydroxyphenylacetic Acid↗

PubMed1999

Pemphigus vulgaris and pemphigus foliaceus antibodies are pathogenic in plasminogen activator knockout mice.

Previous studies have suggested that urokinase plasminogen activator is required for blister formation in pemphigus vulgaris and pemphigus foliaceus. Other studies, however, have shown that downregulation of plasminogen activator does not inhibit blisters induced by pemphigus immunoglobulin G. To eliminate the possibility that small amounts of urokinase plasminogen activator might be sufficient for blister formation, we passively transferred pemphigus immunoglobulin G to urokinase plasminogen activator knockout neonatal mice. Pemphigus foliaceus and pemphigus vulgaris immunoglobulin G caused gross blisters and acantholysis in the superficial and suprabasal epidermis, respectively, to the same degree in knockout and control mice, demonstrating that urokinase plasminogen activator is not absolutely required for antibody-induced blisters. Some studies have shown elevated tissue-type plasminogen activator in pemphigus lesions. Tissue-type plasminogen activator, however, is not necessary for blister formation, because pemphigus foliaceus and pemphigus vulgaris immunoglobulin G caused blisters to the same degree in tissue-type plasminogen activator knockout and control mice. To rule out that one plasminogen activator might compensate for the other in the knockout mice, we bred urokinase plasminogen activator, tissue-type plasminogen activator double knockouts. After passive transfer of pemphigus foliaceus and pemphigus vulgaris immunoglobulin G these mice blistered to the same degree as the single knockout and control mice, and histology indicated blisters at the expected level of the epidermis. These data definitively demonstrate that plasminogen activator is not necessary for pemphigus immunoglobulin G to induce acantholysis in the neonatal mouse model of pemphigus.

Animals↗

PubMed2001

Nitric oxide synthase gene knockout mice do not become hypertensive during pregnancy.

OBJECTIVE: The purpose of this study was to test whether omitting the vasodilator nitric oxide that is derived from any 1 of the 3 isoforms of nitric oxide synthase results in hypertension during pregnancy. STUDY DESIGN: We measured systolic blood pressure before, during, and after pregnancy using an automated tail cuff method in 3 mutant (gene knockout) mouse strains in which the gene for neuronal nitric oxide, inducible nitric oxide, or endothelial nitric oxide was disrupted by gene targeting. RESULTS: In neuronal nitric oxide gene knockout mice (n = 10), blood pressure was 100 +/- 3 mm Hg, not significantly different from 101 +/- 3 mm Hg in matched wild-type control mice (n = 10). Pregnancy did not change blood pressure or heart rate in either group. In inducible nitric oxide gene knockout mice (n = 9), blood pressure was 110 +/- 3 mm Hg, the same as in the wild-type control mice (110 +/- 2 mm Hg; n = 14). Blood pressure was unaffected by pregnancy in either group of mice. However, heart rate was significantly less in knockout mice (647 +/- 11 beats/min vs 666 +/- 9 beats/min; P <.005); this difference persisted through pregnancy. In endothelial nitric oxide gene knockout mice (n = 8), blood pressure was higher before pregnancy (114 +/- 4 mm Hg vs 103 +/- 4 mm Hg; P <.05) than in wild-type control mice (n = 9), but this difference disappeared during pregnancy, returning only after delivery. Heart rates were not different before pregnancy and were unaffected by pregnancy. CONCLUSION: There was no apparent increase in systolic blood pressure in any of the 3 nitric oxide synthase gene knockout strains during pregnancy compared to the wild-type control mice. This suggests that, at least in the mouse, genetic deficiency of any 1 isoform of nitric oxide synthase does not result in pregnancy-induced hypertension.

Animals↗

PubMed2001

Methionine adenosyltransferase 1A knockout mice are predisposed to liver injury and exhibit increased expression of genes involved in proliferation.

Liver-specific and nonliver-specific methionine adenosyltransferases (MATs) are products of two genes, MAT1A and MAT2A, respectively, that catalyze the formation of S-adenosylmethionine (AdoMet), the principal biological methyl donor. Mature liver expresses MAT1A, whereas MAT2A is expressed in extrahepatic tissues and is induced during liver growth and dedifferentiation. To examine the influence of MAT1A on hepatic growth, we studied the effects of a targeted disruption of the murine MAT1A gene. MAT1A mRNA and protein levels were absent in homozygous knockout mice. At 3 months, plasma methionine level increased 776% in knockouts. Hepatic AdoMet and glutathione levels were reduced by 74 and 40%, respectively, whereas S-adenosylhomocysteine, methylthioadenosine, and global DNA methylation were unchanged. The body weight of 3-month-old knockout mice was unchanged from wild-type littermates, but the liver weight was increased 40%. The Affymetrix genechip system and Northern and Western blot analyses were used to analyze differential expression of genes. The expression of many acute phase-response and inflammatory markers, including orosomucoid, amyloid, metallothionein, Fas antigen, and growth-related genes, including early growth response 1 and proliferating cell nuclear antigen, is increased in the knockout animal. At 3 months, knockout mice are more susceptible to choline-deficient diet-induced fatty liver. At 8 months, knockout mice developed spontaneous macrovesicular steatosis and predominantly periportal mononuclear cell infiltration. Thus, absence of MAT1A resulted in a liver that is more susceptible to injury, expresses markers of an acute phase response, and displays increased proliferation.

Animals↗

PubMed1999

Assignment of muscarinic receptor subtypes mediating G-protein modulation of Ca(2+) channels by using knockout mice.

There are five known subtypes of muscarinic receptors (M(1)-M(5)). We have used knockout mice lacking the M(1), M(2), or M(4) receptors to determine which subtypes mediate modulation of voltage-gated Ca(2+) channels in mouse sympathetic neurons. Muscarinic agonists modulate N- and L-type Ca(2+) channels in these neurons through two distinct G-protein-mediated mechanisms. One pathway is fast and membrane-delimited and inhibits N- and P/Q-type channels by shifting their activation to more depolarized potentials. The other is slow and voltage-independent and uses a diffusible cytoplasmic messenger to inhibit both Ca(2+) channel types. Using patch-clamp methods on acutely dissociated sympathetic neurons, we isolated each pathway by pharmacological and kinetic means and found that each one is nearly absent in a particular knockout mouse. The fast and voltage-dependent pathway is lacking in the M(2) receptor knockout mice; the slow and voltage-independent pathway is absent from the M(1) receptor knockout mice; and neither pathway is affected in the M(4) receptor knockout mice. The knockout effects are clean and are apparently not accompanied by compensatory changes in other muscarinic receptors.

Animals↗

PubMed2000

Impaired bone resorption to prostaglandin E2 in prostaglandin E receptor EP4-knockout mice.

Prostaglandin E(2) (PGE(2)) acts as a potent stimulator of bone resorption. In this study, we first clarified in normal ddy mice the involvement of protein kinase A and induction of matrix metalloproteinases (MMPs) in PGE(2)-induced bone resorption, and then identified PGE receptor subtype(s) mediating this PGE(2) action using mice lacking each subtype (EP1, EP2, EP3, and EP4) of PGE receptor. In calvarial culture obtained from normal ddy mice, both PGE(2) and dibutyryl cyclic AMP (Bt(2)cAMP) stimulated bone resorption and induced MMPs including MMP-2 and MMP-13. Addition of an inhibitor of protein kinase A, H89, or an inhibitor of MMPs, BB94, significantly suppressed bone-resorbing activity induced by PGE(2.) In calvarial culture from EP1-, EP2-, and EP3-knockout mice, PGE(2) stimulated bone resorption to an extent similar to that found in calvaria from the wild-type mice. On the other hand, a marked reduction in bone resorption to PGE(2) was found in the calvarial culture from EP4-knockout mice. The impaired bone resorption to PGE(2) was also detected in long bone cultures from EP4-knockout mice. Bt(2)cAMP greatly stimulated bone resorption similarly in both wild-type and EP4-knockout mice. Induction of MMP-2 and MMP-13 by PGE(2) was greatly impaired in calvarial culture from EP4-knockout mice, but Bt(2)cAMP stimulated MMPs induction similarly in the wild-type and EP4-knockout mice. These findings suggest that PGE(2) stimulates bone resorption by a cAMP-dependent mechanism via the EP4 receptor.

Animals↗

PubMed2003

Natural killer cells and B lymphocytes in L-selectin and Mac-1/LFA-1 knockout mice: marker-dependent, but not cell lineage-dependent changes in the spleen and bone marrow.

The majority of B lymphocytes, virgin T lymphocytes and a subpopulation of memory T cells express the addressin, L-selectin. Natural killer (NK) cells in rodents and humans also express L-selectin. We have shown that a similar proportion (40%) of NK cells in mouse spleen also express the integrin, CD18Mac-1, and moreover, that NK cells express both the addressin and the integrin constitutively. It was the aim of the present study to quantify, in knock-out mice deficient for either the L-selectin addressin, or the CD18:Mac-1/LFA-1 integrins, NK cells and B cells in both the spleen and their bone marrow birth site. These cells, in both organs, were immunophenotypically stained with FITC-conjugated anti-NK1.1 (to identify NK cells), and FITC-conjugated anti-mouse B220 (to identify B lymphocytes) and subjected to flow-cytometric analysis using a FACScan equipped with a doublet discrimination module. From the known total organ (spleen, femurs) cellularity, obtained by means of an electronic cell counter, at the time of extraction of each organ, the absolute numbers of NK cells and B lymphocytes from each mouse were obtained. The results revealed that there are significantly more NK cells and B lymphocytes in the spleens of CD18:Mac-1/LFA-1 knockout mice than in control (same strain) mice. Moreover, in L-selectin knockout mice spleens, NK cells and B lymphocytes were elevated by 26.2% and 17.8% respectively. NK cells and B lymphocytes in the bone marrow of the integrin knockout showed no difference from control, however, both cell types in the bone marrow of the L-selectin knockout mice fell to only 3/4 their control levels. Collectively, the results demonstrated that there are organ-specific, but not cell lineage-specific differences in the absolute numbers of NK cells and B lymphocytes, in integrin-deficient (CD18:Mac-1/LFA-1 knockout) mice and addressin-deficient (L-selectin knockout) mice.

Animals↗

PubMed2004

Gene expression profiling of p53(+/-) knockout and wild-type mice following diethylstilbestrol administration.

Studies with clastogenic carcinogen diethylstilbestrol (DES) resulted in a broad of spectrum of toxic and carcinogenic effects in humans and rodents, but the cellular and molecular mechanism(s) by which it induces cancer is not clear. To identify putative genetic targets for p53 in vivo, we applied the cDNA macroarray gene expression profiles associated with apoptosis by comparing p53+/- knockout mice and wild-type mice on the kidney and uterus of female mice. p53+/- knockout mice and wild-type mice were treated with DES (500 micromole kg(-1)) or vehicle i.p once daily for 4 days. Total RNAs were obtained from kidney and uterus of both control and DES-treated. The signal intensities of individual gene spots on the membrane were quantified and normalized to the expression level of the GAPDH gene as an internal control. Our results demonstrated that 16 genes; bad, bax, bcl-2, bcl-w, bcl-x, caspase-3, caspase-7, caspase-8, c-myc, E124, GADD45, mdm2, NKkappab1, p53, p21, Rb and trail were up-regulated and six genes; caspase-1, caspase-2, DR5, E2F1, FasL and iNOS did not changed in response to DES treatment in wild-type mice compared to p53+/- knockout mice. Most genes are involved in cell cycle regulation, signal transduction, apoptosis, or transcription. The greatest changes were seen in bad, bcl-x, mdm2, p53 and p21 gene expression in wild-type mice compared to p53+/- knockout mice. In comparing p53 and p21 gene expression in wild-type mice and p53+/- knockout mice, there was an 4.4-fold vs. 1.8-fold; 8-fold vs. 5.2-fold for kidney and 16-fold vs. 5.5-fold; 2.1-fold vs. 8.3-fold for uterus samples increase in induction (respectively). RT-PCR and densitometric analysis was used to confirm the biggest changes of p21, p53 and bax genes. Using this approach, we have identified apoptosis associated genes regulated in response to DES and have revealed putative differences between the isogenic parent strain and p53+/- knockout mice, which will contribute to a better understanding of toxicity/carcinogenicity mechanisms in this model.

Analysis of Variance↗

PubMed2003

In vitro metabolism of styrene to styrene oxide in liver and lung of Cyp2E1 knockout mice.

Styrene is a widely used chemical. In mice it is both hepatotoxic and pneumotoxic, and this toxicity is thought to be associated with its metabolism to styrene oxide. In vitro studies by several investigators suggest that this bioactivation in mice is primarily due to CYP2E1 and CYP2F2. However, in vivo studies demonstrate that CYP2E1 knockout mice can metabolize styrene to a similar extent as the wild-type mice. The current studies compared the in vitro metabolism of styrene by hepatic and pulmonary microsomes from CYP2E1 knockout and wild-type mice. There was no difference in the hepatic microsomal metabolism of styrene to styrene oxide between the two strains. The metabolism of styrene was lower in the lungs of the knockout mice than in the wild-type. Chemical inhibitors were used to ascertain the contributions made by various cytochromes P-450: imipramine for CYP2C, alpha -methylbenzylaminobenzotriazole for CYP2B, alpha -naphthoflavone for CYP1A, 5-phenyl-1-pentyne for CYP2F2, and diethyldithiocar-bamate for CYP2E1. The data indicate that CYP2E1 and CYP2F2 may be important in wild-type mice, but they do not clearly indicate what cytochromes P-450 are responsible for the metab-olism in the knockout mice. Inhibition of styrene metabolism in the knockout mice by diethyl-dithiocarbamate indicates this inhibitor is not completely selective for CYP2E1. These in vitro data support the in vivo finding of styrene metabolism in CYP2E1 knockout mice and indicate that other enzymes are contributing to styrene metabolism in these mice.

Animals↗

PubMed2001

Cytochrome P450 and antioxidant activity in interleukin-6 knockout mice after induction of the acute-phase response.

Hepatic cytochrome P450 (CYP) expression and antioxidant activity have been shown to decrease following endotoxin (lipopolysaccharide [LPS]) or proinflammatory cytokine administration. Using mice deficient in interleukin-6 (IL-6), the role of IL-6 in the regulation of hepatic CYP activity, glutathione (GSH) metabolism, and catalase (CAT) activity was analyzed after LPS administration. Administration of LPS produced comparable decreases in hepatic CYP3A activity in WT B6x129 (WT) mice and IL-6 knockout mice. No decrease was observed for CYP2D9 activity after LPS administration in either WT or IL-6 knockout mice. LPS administration significantly increased hepatic and renal CYP2E1 and CYP4A activity in WT mice, with no effect in IL-6 knockout mice. CYP2A12 activity increased in IL-6 knockout, mice with no change in WT mice after LPS administration. LPS administration had no significant effect on hepatic GSH reductase, GST peroxidase, GSH-S-transferase (GST), or total GSH in either WT or IL-6 knockout. However, hepatic CAT activity was significantly reduced in WT mice after LPS administration, with no effect in IL-6 knockout mice. These results support IL-6 as a critical mediator of the effects of LPS on specific hepatic and renal CYP activities and hepatic CAT activity.

Acute-Phase Reaction↗

PubMed2006

Detection of NaCl and KCl in TRPV1 knockout mice.

Both amiloride-sensitive and -insensitive mechanisms contribute to NaCl taste transduction. The amiloride-sensitive mechanism relies on the epithelial Na(+) channel ENaC, which is widely expressed on the apical membrane of fungiform taste cells. The amiloride-insensitive mechanism, which predominates in circumvallate and foliate taste buds, was recently reported to involve a variant of the nonselective cation channel TRPV1. We performed 2-bottle preference and threshold experiments with TRPV1 knockout mice and wild-type (C57BL/6J) controls to test for NaCl preference and detection thresholds in the presence and absence of amiloride. Surprisingly, TRPV1 knockout mice not only detected NaCl in the presence of amiloride but they preferred NaCl over water at concentrations avoided by the wild-type mice. NaCl detection thresholds were between 2 and 3 mM for both genotypes. Amiloride increased the detection thresholds of wild-type mice but not knockout mice. The knockout mice also preferred 100 mM KCl compared with wild-type controls, suggesting that TRPV1 receptors may mediate a general aversive response to salts. Analyses of consumption data also revealed that TRPV1 knockout mice ingested more of the NaCl, with and without amiloride, and KCl solutions than the wild-type mice. However, comparisons of preference ratios and consumption volumes indicated that both wild-type and TRPV1 knockout mice avoided citric acid in quite a similar manner, suggesting that TRPV1 receptors do not mediate the detection of citric acid. These data, taken together, suggest that additional mechanisms must contribute to the amiloride-insensitive NaCl response.

Amiloride↗

PubMed1995

The role of transgenic knockout models in defining the pathogenesis of viral heart disease.

The pathogenesis of viral myocarditis involves contributions from the virus, the immune system and myocytes. In defining the molecular contributions in the disease process, modulations of the components of the immune system through transgenic knockout models provide useful insights. Advantages of the transgenic knockout models are that they allow biological evaluation of the importance of a particular molecule in the physiological context of an intact organism. Furthermore, the techniques of transgenic knockout models are now standardized, even though they are still technically challenging and time consuming. An example in myocarditis is the IRF-1 knockout mouse, where there is a complete absence of the inducible form of nitric oxide synthetase in the tissues. These animals are exquisitely sensitive to coxsackieviral infection, with extremely high mortality. On the other hand, CD4 knockouts appear to still have myocarditis in an autoimmune myocarditis model, while p56lck knockouts (the T-cell tyrosine kinase signalling molecule) appears to be free of viral myocarditis. These elegant systems of molecular manipu-lation should allow us unique insights into the pathogenesis of myocarditis.

Animals↗

PubMed2001

Lack of change in the levels of liver and kidney cytochrome P-450 isozymes in p53+/- knockout mice treated with N-butyl-N-(4-hydroxybutyl)nitrosamine.

We have previously shown that p53(+/-) knockout mice are highly sensitive to urinary bladder carcinogenesis induced by N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) in spite of a lack of effects of p53 heterozygosity on N-butyl-N-(3-carboxypropyl)nitrosamine (BCPN) excretion in urine. To determine the influence of p53 deficiency on in vitro formation of BCPN, mutagenicity of BBN and BCPN and levels of several cytochrome P450 (CYP) isozymes, groups of five p53(+/-) knockout and wild-type mice (littermates), as well as animals of the C57BL/6 parental strain, were administered 0.025% BBN in their drinking water for 4 weeks. The livers and kidneys were then used for analyses of BBN metabolism, western immunoblotting and Ames liquid incubation. BBN treatment caused a slight decrease in BCPN formation in the livers of C57BL/6 mice, but there was no significant difference between p53 knockout, wild-type and C57BL/6 mice. In kidney BCPN formation in p53 knockout mice was 33-46% less than that in their wild-type counterparts. Using anti-rat CYP antibodies, CYP1A2, 2B9/10, 2E1 and 3A11/13 were constitutively detected in liver microsomes and CYP2E1 and 3A11/13 in the kidney. Densitometric determination of these CYP proteins revealed no significant variation in levels detected in both tissues among the four groups of mice. BBN and BCPN were not mutagenic for Salmonella typhimurium TA100 in either the absence or presence of liver S9 from untreated mice and rats and from p53 knockout mice treated with BBN. In conclusion, p53 deficiency and BBN had no enhancing effects on metabolism of BBN to BCPN and expression of the CYP isozymes typically responsible for activation of environmental carcinogens, including both of the N-nitrosamines tested, and their mutagenicity, indicating that the high susceptibility of p53(+/-) knockout mice is not attributable to metabolic activation in liver and kidney by CYP isozymes or urinary excretion of BCPN.

Animals↗

PubMed2004

Leukocyte subpopulations in the uteri of leukemia inhibitory factor knockout mice during early pregnancy.

Leukemia inhibitory factor (LIF) is transiently expressed on Day (D) 1 of pregnancy by the uterine epithelium and on D4 specifically by the glandular epithelium. The Lif knockout female mice are infertile because of uterine defects that affect embryo implantation, but pregnancy can be rescued in these mice by injections of LIF on D4 of pregnancy. Many of the specific actions of LIF in the uterus are unknown, especially with regard to uterine cell biology. Leukocytes, such as macrophages, natural killer (NK) cells, and eosinophils, are present in the pregnant uterus and are thought to be beneficial, because alterations in their proportions can adversely affect pregnancy. Immunocytochemistry and cell counting were used to compare the distributions and dynamics of leukocyte subpopulations in wild-type and Lif knockout mice. The percentage of macrophages was reduced by more than half in the Lif knockout mice on D3 of pregnancy, and their distribution was disrupted, suggesting that LIF is a chemokine for these cells. The NK cells were detected as early as D3 of pregnancy, but the Lif knockout mice had double the percentage of NK cells compared to wild-type mice at this time, indicating that LIF restricts the migration of NK cells to the uterus. The Lif knockout mice also had significantly higher percentages of eosinophils in the outer stroma on D3, and in the midstroma on D4, of pregnancy, suggesting that LIF also may restrict eosinophil migration to the uterus. These alterations of the uterine leukocyte subpopulations in Lif knockout mice may disrupt pregnancy and contribute to failure of implantation.

Animals↗

PubMed2000

The cause of infertility of male c-ros tyrosine kinase receptor knockout mice.

Male homozygous transgenic c-ros knockout mice are sterile by natural mating, lack a part of their epididymis, and the epididymal sperm exhibit tail angulation in vivo and in vitro. To ascertain if this abnormal tail form caused the infertility, the number and nature of sperm in the tract of females mated to knockout and wild-type mice were determined. Percentage motility and numbers of sperm in the uterus 1 h after mating were similar between genotypes. The majority of the uterine sperm from the wild-type males had straight flagella, whereas 46-86% of knockout sperm were bent at the cytoplasmic droplet even when motile. Motile knockout sperm showed a 54 and 37% reduction in the straightline and curvilinear velocities compared with straight wild-type sperm. Sequential flushings of the oviduct 4 h after mating with the wild-type males contained sperm: 591 +/- 119 free, 371 +/- 70 loosely, and 122 +/- 47 tightly bound to the epithelium, but no knockout sperm were recovered from the oviduct or observed within the uterotubal junction in tissue sections. The infertility of c-ros knockout male mice can be explained by the sperm's inability to enter the oviduct, as a result of their bent tails forming the entangled sperm mass and their compromised flagellar vigor within the uterus.

Animals↗

PubMed2001

Sensory processing in the deep spinal dorsal horn of neurokinin-1 receptor knockout mice.

BACKGROUND: The neurokinin-1 receptor and its primary ligand, substance P, are widely recognized as contributing to the spinal processing of nociceptive stimuli, yet the specific function of the neurokinin-1 receptor remains unclear. METHODS: To better clarify these functions, the authors examined the neurophysiologic responses of L4-L5 neurons in the deep dorsal horn to acute mechanical, thermal, and electrical stimuli in knockout and wild-type mice. In addition, the capacity of knockout and wild-type mice to show wind-up to repeated C-fiber stimuli and to show sensitization after cutaneous mustard oil was assessed. RESULTS: A total of 68 nociceptive neurons (35 in knockout, 33 in wild type) in laminae III-V were studied. No differences in the acute responses of neurons in knockout and wild-type mice to graded mechanical, thermal, or electrical stimuli or in the acute responses to mustard oil were observed. However, wind-up to repeated electrical stimulation at C-fiber intensity was significantly attenuated in the knockout mice compared with wild type controls. In addition, mustard oil-induced mechanical hypersensitivity was significantly reduced in the knockout mice. CONCLUSIONS: These results indicate that neurokinin-1 receptors do not play a significant role in the responses of nociceptive neurons in the deep spinal dorsal horn to acute noxious mechanical, thermal, electrical, or chemical stimuli. On the other hand, neurokinin-1 receptors are critical for the central hyperexcitability that is observed in these neurons with repeated C-fiber inputs and to the central sensitization induced by topical mustard oil application.

Animals↗

PubMed2003

Myocardial dysfunction and male mortality in peroxisome proliferator-activated receptor alpha knockout mice overexpressing lipoprotein lipase in muscle.

Free fatty acids (FFA) are liberated from triglyceride-rich lipoproteins by lipoprotein lipase (LPL) and are considered to be a principal energy source for the heart. The peroxisome proliferator-activated receptor alpha (PPARalpha) is a key regulator of FFA catabolism. To investigate its role in cardiac muscle metabolism, transgenic mice overexpressing LPL in skeletal and cardiac muscle were bred on a PPARalpha knockout background. Fifty-five percent of male animals lacking PPARalpha and overexpressing LPL died within 4 months after birth. In contrast, females of this genotype stayed alive. Deceased animals exhibited cardiopulmonary congestion but had no increase of neutral lipids in the heart. Changes in plasma glucose, FFA, lactate, and triglycerides did not clearly account for gender-specific differences in mortality; however, they indicated a critical role for PPARalpha during fasting. Analysis of cardiac function revealed that in isolated perfused hearts, left ventricular developed pressure (a measure of contractility) was markedly lower in PPARalpha knockout mice overexpressing LPL compared with controls. Glucose uptake of isolated perfused hearts was significantly higher in PPARalpha knockout mice with both normal or increased LPL expression. However, uptake of FFA was not different among genotypes. In contrast, fasted FFA levels were significantly lower in cardiac muscle of PPARalpha knockout mice with normal LPL expression (-26%) and PPARalpha knockout mice overexpressing LPL (-38%) compared with controls. Our results indicate a critical role for PPARalpha in myocardial pump function and suggest that mouse models combining different genetic effects such as PPARalpha knockout mice overexpressing muscle LPL may be useful to study cardiomyopathies.

Animals↗

PubMed2002

Invasion of melanoma in double knockout mice lacking tenascin-X and tenascin-C.

The roles of extracellular matrix glycoproteins belonging to the tenascin family in the regulation of tumor cell proliferation, invasion, and metastasis are not known. To address this issue, we generated tenascin-X (TNX) and tenascin-C (TNC) double knockout mice and compared findings in these mice with those in single knockout (TNX + / + TNC - / - and TNX - / - TNC + / +) mice. We investigated the proliferation and invasion of B16-BL6 melanoma cells after these cells had been injected into the footpads of mice in each group. The primary tumor size and invasion to the ankle adjacent to the primary tumor site were examined at 35 days after injection of the melanoma cells. The primary tumor size in TNX - / - TNC + / + mice was significantly larger than that in wild-type mice, but those of TNX + / + TNC - / - and double knockout mice were comparable to that in the wild-type mice. On the other hand, invasion to the ankle was obviously promoted in TNX - / - TNC + / + and double knockout mice compared with that in the wild-type mice, but invasion to the ankle in TNX + / + TNC - / - mice was only slightly promoted. Gelatin zymography confirmed increased matrix metalloproteinase (MMP)-9 activity in the dorsal skin of TNX - / - TNC + / +, TNX + / + TNC - / - and double knockout mice. However, the amounts of MMP-9 mRNA in the dorsal skins of all mice were almost the same, indicating that the increased activity of MMP-9 in the single and double knockout mice is regulated at the MMP-9 processing level. These results indicate that MMP-9 is activated in all TN-deficient mice, but that TNX exerts a greater effect on tumor invasion than does TNC.

Animals↗