Altered Sleep Homeostasis in Rev-erbα Knockout Mice.

STUDY OBJECTIVES: The nuclear receptor REV-ERBα is a potent, constitutive transcriptional repressor critical for the regulation of key circadian and metabolic genes. Recently, REV-ERBα's involvement in learning, neurogenesis, mood, and dopamine turnover was demonstrated suggesting a specific role in central nervous system functioning. We have previously shown that the brain expression of several core clock genes, including Rev-erbα, is modulated by sleep loss. We here test the consequences of a loss of REV-ERBα on the homeostatic regulation of sleep. METHODS: EEG/EMG signals were recorded in Rev-erbα knockout (KO) mice and their wild type (WT) littermates during baseline, sleep deprivation, and recovery. Cortical gene expression measurements after sleep deprivation were contrasted to baseline. RESULTS: Although baseline sleep/wake duration was remarkably similar, KO mice showed an advance of the sleep/wake distribution relative to the light-dark cycle. After sleep onset in baseline and after sleep deprivation, both EEG delta power (1-4 Hz) and sleep consolidation were reduced in KO mice indicating a slower increase of homeostatic sleep need during wakefulness. This slower increase might relate to the smaller increase in theta and gamma power observed in the waking EEG prior to sleep onset under both conditions. Indeed, the increased theta activity during wakefulness predicted delta power in subsequent NREM sleep. Lack of Rev-erbα increased Bmal1, Npas2, Clock, and Fabp7 expression, confirming the direct regulation of these genes by REV-ERBα also in the brain. CONCLUSIONS: Our results add further proof to the notion that clock genes are involved in sleep homeostasis. Because accumulating evidence directly links REV-ERBα to dopamine signaling the altered homeostatic regulation of sleep reported here are discussed in that context.

The role of PPARgamma in cartilage growth and development using cartilage-specific PPARgamma knockout mice

Le cartilage est un tissu conjonctif composé d’une seule sorte de cellule nommée chondrocytes. Ce tissu offre une fondation pour la formation des os. Les os longs se développent par l'ossification endochondral. Ce processus implique la coordination entre la prolifération, la différenciation et l'apoptose des chondrocytes, et résulte au remplacement du cartilage par l'os. Des anomalies au niveau du squelette et des défauts liés à l’âge tels que l’arthrose (OA) apparaissent lorsqu’il y a une perturbation dans l’équilibre du processus de développement. À ce jour, les mécanismes exacts contrôlant la fonction et le comportement des chondrocytes pendant la croissance et le développement du cartilage sont inconnus. Le récepteur activateur de la prolifération des peroxysomes (PPAR) gamma est un facteur de transcription impliqué dans l'homéostasie des lipides. Plus récemment, son implication a aussi été suggérée dans l'homéostasie osseuse. Cependant, le rôle de PPARγ in vivo dans la croissance et le développement du cartilage est inconnu. Donc, pour la première fois, cette étude examine le rôle spécifique de PPARγ in vivo dans la croissance et le développement du cartilage. Les souris utilisées pour l’étude avaient une délétion conditionnelle au cartilage du gène PPARγ. Ces dernières ont été générées en employant le système LoxP/Cre. Les analyses des souris ayant une délétion au PPARγ aux stades embryonnaire et adulte démontrent une réduction de la croissance des os longs, une diminution des dépôts de calcium dans l’os, de la densité osseuse et de la vascularisation, un délai dans l’ossification primaire et secondaire, une diminution cellulaire, une perte d’organisation colonnaire et une diminution des zones hypertrophiques, une désorganisation des plaques de croissance et des chondrocytes déformés. De plus, la prolifération et la différenciation des chondrocytes sont anormales. Les chondrocytes et les explants isolés du cartilage mutant démontrent une expression réduite du facteur de croissance endothélial vasculaire (VEGF)-A et des éléments de production de la matrice extracellulaire. Une augmentation de l’expression de la métalloprotéinase matricielle (MMP)-13 est aussi observée. Dans les souris âgées ayant une délétion au PPARγ, y est aussi noté des phénotypes qui ressemblent à ceux de l’OA tel que la dégradation du cartilage et l'inflammation de la membrane synoviale, ainsi qu’une augmentation de l’expression de MMP-13 et des néoépitopes générés par les MMPs. Nos résultats démontrent que le PPARγ est nécessaire pour le développement et l’homéostasie du squelette. PPARγ est un régulateur essentiel pour la physiologie du cartilage durant les stades de croissance, de développement et de vieillissement.

Increased vascular contractility and oxidative stress in beta(2)-adrenoceptor knockout mice: the role of NADPH oxidase

Background/Aims: beta(2)-adrenoceptor (beta(2)-AR) activation induces smooth muscle relaxation and endothelium-derived nitric oxide (NO) release. However, whether endogenous basal beta(2)-AR activity controls vascular redox status and NO bioavailability is unclear. Thus, we aimed to evaluate vascular reactivity in mice lacking functional beta(2)-AR (beta 2KO), focusing on the role of NO and superoxide anion. Methods and Results: Isolated thoracic aortas from beta 2KO and wild-type mice (WT) were studied. beta 2KO aortas exhibited an enhanced contractile response to phenylephrine compared to WT. Endothelial removal and L-NAME incubation increased phenylephrine-induced contraction, abolishing the differences between beta 2KO and WT mice. Basal NO availability was reduced in aortas from beta 2KO mice. Incubation of beta 2KO aortas with superoxide dismutase or NADPH inhibitor apocynin restored the enhanced contractile response to phenylephrine to WT levels. beta 2KO aortas exhibited oxidative stress detected by enhanced dihydroethidium fluorescence, which was normalized by apocynin. Protein expression of eNOS was reduced, while p47(phox) expression was enhanced in beta 2KO aortas. Conclusions: The present results demonstrate for the first time that enhanced NADPH-derived superoxide anion production is associated with reduced NO bioavailability in aortas of beta 2KO mice. This study extends the knowledge of the relevance of the endogenous activity of beta(2)-AR to the maintenance of the vascular physiology. Copyright (C) 2012 S. Karger AG, Basel

Sensitivity to endothelin-1 is decreased in isolated livers of endothelial constitutive nitric oxide synthase knockout mice

ABSTRACT: BACKGROUND: Hepatic sinusoidal resistance is regulated by vasoactive factors including endothelin-1 (ET-1) and nitric oxide (NO). In the absence of NO, vasoconstrictor response to endothelin is expected to predominate. Therefore, we hypothesized sensitivity to endothelin to be increased in mice lacking the endothelial cell NO synthase gene. Response of vascular resistance to endothelin was assessed in the in situ perfused liver of endothelial constitutive nitric oxide synthase (ecNOS) knockout and wild type mice. Livers were also harvested for RNA and protein isolation for quantitative PCR and Western blotting, respectively. The expression of endothelin receptors, isoenzymes of NO synthase, heme-oxygenase and adrenomedullin was quantified. RESULTS: Endothelin increased hepatic vascular resistance in a dose-dependent manner in both strains; however, this increase was significantly less in ecNOS knockout mice at physiologic concentrations. Expression of heme-oxygenases and adrenomedullin was similar in both groups, whereas inducible nitric oxide synthase (iNOS) protein was not detectable in either strain. mRNA levels of pre-pro-endothelin-1 and ETB receptor were comparable in both strains, while mRNA for ETA receptor was decreased in ecNOS knockouts. CONCLUSION: Livers of ecNOS knockout mice have a decreased sensitivity to endothelin at physiologic concentrations; this is associated with a decreased expression of ETA receptors, but not with other factors, such as iNOS, ETB receptors, adrenomedullin or heme-oxygenase. Further studies targeting adaptive changes in ETA receptor distribution and/or intracellular signaling downstream of the receptor are indicated.

Phenotype of capillaries in skeletal muscle of nNOS-knockout mice

Because neuronal nitric oxide synthase (nNOS) has a well-known impact on arteriolar blood flow in skeletal muscle, we compared the ultrastructure and the hemodynamics of/in the ensuing capillaries in the extensor digitorum longus (EDL) muscle of male nNOS-knockout (KO) mice and wild-type (WT) littermates. The capillary-to-fiber (C/F) ratio (-9.1%) was lower (P ≤ 0.05) in the nNOS-KO mice than in the WT mice, whereas the mean cross-sectional fiber area (-7.8%) and the capillary density (-3.1%) varied only nonsignificantly (P > 0.05). Morphometrical estimation of the area occupied by the capillaries as well as the volume and surface densities of the subcellular compartments differed nonsignificantly (P > 0.05) between the two strains. Intravital microscopy revealed neither the capillary diameter (+3% in nNOS-KO mice vs. WT mice) nor the mean velocity of red blood cells in EDL muscle (+25% in nNOS-KO mice vs. WT mice) to significantly vary (P > 0.05) between the two strains. The calculated shear stress in the capillaries was likewise nonsignificantly different (3.8 ± 2.2 dyn/cm² in nNOS-KO mice and 2.1 ± 2.2 dyn/cm² in WT mice; P > 0.05). The mRNA levels of vascular endothelial growth factor (VEGF)-A were lower in the EDL muscle of nNOS-KO mice than in the WT littermates (-37%; P ≤ 0.05), whereas mRNA levels of VEGF receptor-2 (VEGFR-2) (-11%), hypoxia inducible factor-1α (+9%), fibroblast growth factor-2 (-14%), and thrombospondin-1 (-10%) differed nonsignificantly (P > 0.05). Our findings support the contention that VEGF-A mRNA expression and C/F-ratio but not the ultrastructure or the hemodynamics of/in capillaries in skeletal muscle at basal conditions depend on the expression of nNOS.

Disruption of estrogen signaling does not prevent progesterone action in the estrogen receptor α knockout mouse uterus

Estrogen is known to increase progesterone receptor (PR) levels in the wild-type mouse uterus, and this estrogen induction was thought to be important for progesterone action through the PR. The estrogen receptor α knockout (ERKO) mouse uterus was observed to express PR mRNA that cannot be induced by estrogen. Progesterone action was characterized to determine whether it was diminished in ERKO mice. The PR protein is present in the ERKO uterus at 60% of the level measured in a wild-type uterus. The PR-A and PR-B isoforms are both detected on Western blot, and the ratio of isoforms is the same in both genotypes. Although the level of PR is reduced in the ERKO uterus, the receptor level is sufficient to induce genomic responses, since both calcitonin and amphiregulin mRNAs were increased after progesterone treatment. Finally, the ERKO uterus can be induced to undergo a progesterone-dependent decidual response. Surprisingly, the decidual response is estrogen independent in the ERKO, although it remains estrogen dependent in a wild type. These results indicate that estrogen receptor α modulation of PR levels is not necessary for expression of the PR or genomic and physiologic responses to progesterone in the ERKO uterus.

Responses in the brain of estrogen receptor α-disrupted mice

These studies sought to determine if neurons in the estrogen receptor-α knockout (ERαKO) mouse brain concentrated 16α-[125I]iodo-11β-methoxy-17β-estradiol (125I-estrogen), and if so, whether estrogen binding augmented the expression of progesterone receptor (PR) mRNA. Mice were injected with 125I-estrogen and cryostat sections thaw mounted onto emulsion-coated slides. After 30–90 days of exposure, cells with a nuclear uptake and retention of 125I-estrogen were observed in a number of ERαKO mouse brain regions including the preoptic nucleus and arcuate nucleus of the hypothalamus, bed nucleus of the stria terminalis, and amygdala, although the number of labeled cells and intensity of nuclear concentration was markedly attenuated when compared with wild-type littermates. Competition studies with excess 17β-estradiol, diethylstilbestrol, or moxestrol, but not with R5020 or dihydrotestosterone, prevented the nuclear concentration of 125I-estrogen. To determine if the low level of estrogen binding was capable of regulating gene expression, in situ hybridization was used to evaluate PR mRNA in the brain. ERαKO and wild-type mice were ovariectomized and treated with vehicle or 17β-estradiol, and brains were sectioned and hybridized with a PR cRNA probe. Analysis of hybridization signal revealed a similar, low level of PR mRNA in ovariectomized wild-type and homozygous mice, and a marked increase in expression after treatment of ovariectomized animals with 17β-estradiol, with the level of hybridization signal being significantly higher in wild-type animals when compared with ERαKO mice. The results demonstrate that estrogen binds in the ERαKO brain and is capable of modulating PR gene expression, thus supporting the presence and functionality of a nonclassical estrogen receptor.

Alternatively spliced forms in the cytoplasmic domain of the human growth hormone (GH) receptor regulate its ability to generate a soluble GH-binding protein.

The mechanism underlying the generation of soluble growth hormone binding protein (GHBP) probably differs among species. In rats and mice, it involves an alternatively spliced mRNA, whereas in rabbits, it involves limited proteolysis of the membrane-bound growth hormone receptor (GHR). In humans, this latter mechanism is favored, as no transcript coding for a soluble GHR has been detected so far. To test this hypothesis, we analyzed COS-7 cells transiently expressing the full-length human (h) GHR and observed specific GH-binding activity in the cell supernatants. Concomitantly, an alternatively spliced form in the cytoplasmic domain of GHR, hGHR-tr, was isolated from several human tissues. hGHR-tr is identical in sequence to hGHR, except for a 26-bp deletion leading to a stop codon at position 280, thereby truncating 97.5% of the intracellular domain of the receptor protein. When compared with hGHR, hGHR-tr showed a significantly increased capacity to generate a soluble GHBP. Interestingly, this alternative transcript is also expressed in liver from rabbits, mice, and rats, suggesting that, in these four species, proteolysis of the corresponding truncated transmembrane GHR is a common mechanism leading to GHBP generation. These findings support the hypothesis that GHBP may at least partly result from alternative splicing of the region encoding the intracellular domain and that the absence of a cytoplasmic domain may be involved in increased release of GHBP.

Efeito das gorduras interesterificadas sobre o desenvolvimento da lesão aterosclerótica em camundongos knockout para o receptor de LDL

Nas últimas décadas, diversos estudos têm demonstrado os efeitos nocivos dos ácidos graxos trans à saúde. Consequentemente, diversas agências reguladoras de saúde e sociedades responsáveis pela elaboração de diretrizes nutricionais recomendaram a redução do consumo desses ácidos graxos. Deste modo, a indústria de alimentos vem adequando seus produtos a fim de substituir os ácidos graxos trans por gorduras interesterificadas, porém seus efeitos sobre o desenvolvimento da aterosclerose não foram ainda totalmente elucidados. Portanto, o objetivo deste estudo foi avaliar o efeito de gorduras interesterificadas contendo principalmente ácido graxo palmítico ou esteárico sobre o desenvolvimento da aterosclerose. Desta forma, camundongos knockout para o receptor de LDL (LDLr-KO) recém-desmamados foram alimentados por 16 semanas com dietas hiperlipídicas (40% do valor calórico total sob forma de gordura) contendo principalmente ácidos graxos poli-insaturados (POLI), trans (TRANS), palmítico (PALM), palmítico interesterificado (PALM INTER), esteárico (ESTEAR) ou esteárico interesterificado (ESTEAR INTER) para determinação de concentrações plasmáticas de colesterol total e triglicérides; perfil de lipoproteínas; conteúdo de lípides (Oil Red O) e colágeno (Picrosirius Red) e infiltrado de macrófagos (imuno-histoquímica) na área de lesão aterosclerótica; expressão e conteúdo proteico de citocinas na aorta; dosagem das citocinas secretadas por macrófagos de peritônio estimulados ou não com lipopolissacarídeo (LPS); efluxo celular de colesterol mediado pela apo-AI e HDL2. Os resultados mostraram que os animais que consumiram a gordura interesterificada contendo ácido palmítico (PALM INTER) desenvolveram importante lesão aterosclerótica em comparação aos grupos PALM, ESTEAR, ESTEAR INTER e POLI, resultados confirmados pelo conteúdo de colágeno na lesão. Apesar do processo de interesterificação não ter alterado as concentrações plasmáticas de lípides, conforme verificado entre os grupos PALM vs PALM INTER e ESTEAR vs ESTEAR INTER, o acúmulo de colesterol na partícula de LDL foi similar entre os grupos PALM INTER e TRANS. Além desse efeito sobre o perfil de lipoproteínas, macrófagos do peritônio de camundongos que consumiram PALM INTER secretaram significativamente mais IL-1beta, IL-6 e MCP-1 em comparação aos demais grupos. Esse efeito pró-inflamatório foi confirmado na aorta, onde se observou maior expressão de TNF-alfa e IL-1beta para o grupo PALM INTER em comparação a PALM. Tal insulto inflamatório foi similar ao provocado por TRANS. Esses efeitos deletérios do PALM INTER podem ser parcialmente atribuídos ao acúmulo de colesterol nos macrófagos, promovido pelo prejuízo no efluxo de colesterol mediado pela apo-AI e HDL2, bem como aumento da expressão de receptores envolvidos na captação de LDL modificada (Olr-1) e diminuição daqueles envolvidos na remoção intracelular de colesterol (Abca1 e Nr1h3) na parede arterial. Como conclusão, as gorduras interesterificadas contendo ácido palmítico favorecem o acúmulo de colesterol nas partículas de LDL e em macrófagos, ativando o processo inflamatório, o que conjuntamente contribuiu para maior desenvolvimento de lesão aterosclerótica

Influence of growth hormone on the craniofacial complex of transgenic mice

Growth hormone (GH) secretion affects bone and cartilage physiology. This study investigated the effect of GH on the size of the craniofacial structures and their angular relationship. Three different models of mice with a genetically altered GH axis were used: GH excess (giant), dwarf GH antagonist (dwarf-Ant), and dwarf GH receptor knockout (dwarf-KO) mice. Each model was compared with the corresponding wild type (Wt). Five craniofacial distances were analysed: craniofacial length, upper face height, mandibular anterior height, mandibular ramus length, and mandibular corpus length. In addition, upper and lower incisor lengths and four angular relationships, nasal bone with cranial base, maxillary plane with cranial base, mandibular plane with cranial base, and the angle of the mandible, were determined. Data were analysed by one-way ANOVA. Craniofacial length, upper face height and mandibular corpus length were significantly increased in the giant mice and significantly reduced in the dwarf mice. Mandibular anterior height and mandibular ramus length were significantly affected in the dwarf-KO mice but not in the giant mice. The length of both the upper and lower incisors was significantly increased and reduced in the giant and dwarf-KO mice, respectively. In addition, the angle of the mandible was significantly increased in the giant mice and significantly reduced in the dwarf mice. It is concluded that GH plays a major role in the growth and development of the craniofacial complex by directly and indirectly modulating the size and the angular relationships of the craniofacial structures, including the incisor teeth.

Leishmania spp. parasite isolation through inoculation of patient biopsy macerates in interferon gamma knockout mice

Isolation of Leishmania parasite and species identification are important for confirmation and to help define the epidemiology of the leishmaniasis. Mice are often used to isolate pathogens, but the most common mouse strains are resistant to infection with parasites from the Leishmania (Viannia) subgenus. In this study we tested the inoculation of interferon gamma knockout (IFNγ KO) mice with biopsy macerates from Leishmania-infected patients to increase the possibility of isolating parasites. Biopsies from twenty five patients with clinical signs of leishmaniasis were taken and tested for the presence of parasites. Immunohistochemical assay (IHC) and conventional histopathology detected the parasite in 88% and 83% of the patients, respectively. Leishmania sp. were isolated in biopsy macerates from 52% of the patients by culture in Grace's insect medium, but 13% of isolates were lost due to contamination. Inoculation of macerates in IFNγ KO mice provides isolation of parasites in 31.8% of the biopsies. Most isolates belong to L. (Viannia) subgenus, as confirmed by PCR, except one that belongs to L. (Leishmania) subgenus. Our preliminary results support the use of IFNγ KO mice to improve the possibility to isolate New World Leishmania species.

Remyelination in experimentally demyelinated connexin 32 KnockOut mice

The aim of this study was to evaluate the role of connexin 32 (Cx 32) during remyelination of the peripheral nervous system, through a local injection of either 0,1% ethidium bromide solution or saline in the sciatic nerve of Cx 32 knockout mice. Euthanasia was performed ranging from 1, 2, 3, 7, 15, 21 to 30 days after injection. Histochemical, immunohistochemical, immunofluorescence and transmission electron microscopical techniques were used to analyze the development of the lesions. Within the sciatic nerves, Schwann cells initially showed signs of intoxication and rejected their sheaths; after seven days, some thin newly formed myelin sheaths with uneven compactness and redundant loops (tomacula) were conspicuous. We concluded that the regeneration of lost myelin sheaths within the PNS followed the pattern already reported for this model in other laboratory species. Therefore, these results suggest that absence of Cx 32 did not interfere with the normal pattern of remyelination in this model in young mice.

Contribution of beta-adrenoceptor subtypes to relaxation of colon and oesophagus and pacemaker activity of ureter in wild-type and beta(3)-adrenoceptor knockout mice

1 The smooth muscle relaxant responses to the mixed beta(3)-, putative beta(4)-adrenoceptor agonist, (-)-CGP 12177 in rat colon are partially resistant to blockade by the beta(3)-adrenoceptor antagonist SR59230A suggesting involvement of beta(3)- and putative beta(4)-adrenoceptors. We now investigated the function of the putative beta(4)-adrenoceptor and other beta-adrenoceptor subtypes in the colon, oesophagus and ureter of wild-type (WT) and beta(3)-adrenoceptor knockout (beta(3)KO) mice. 2 (-)-Noradrenaline and (-)-adrenaline relaxed KCl (30 mM)-precontracted colon mostly through beta(1)-and beta(3)-adrenoceptors to a similar extent and to a minor extent through beta(2)-adrenoceptors. In colon from beta(3)KO mice, (-)-noradrenaline was as potent as in WT mice but the effects were mediated entirely through beta(1)-adrenoceptors. (-)-CGP 12177 relaxed colon from beta(3)KO mice with 2 fold greater potency than in WT mice. The maintenance of potency for (-)-noradrenaline and increase for (-)-CGP 12177 indicate compensatory increases in beta(1)- and putative beta(4)-adrenoceptor function in beta(3)KO mice. 3 In oesophagi precontracted with 1 mu M carbachol, (-)-noradrenaline caused relaxation mainly through beta(1)-and beta(3)-adrenoceptors. (-)-CGP 12177 (2 mu M) relaxed oesophagi from WT by 61.4+/-5.1% and beta(3)KO by 67.3+/-10.1% of the (-)-isoprenaline-evoked relaxation, consistent with mediation through putative beta(4)-adrenoceptors. 4 In ureter, (-)-CGP 12177 (2 mu M) reduced pacemaker activity by 31.1+/-2.3% in WT and 31.3+/-7.5% in beta(3)KO, consistent with mediation through putative beta(4)-adrenoceptors. 5 Relaxation of mouse colon and oesophagus by catecholamines are mediated through beta(1)- and beta(3)- adrenoceptors in WT. The putative beta(4)-adrenoceptor, which presumably is an atypical state of the beta(1)-adrenoceptor, mediates the effects of(-)-CGP 12177 in colon, oesophagus and ureter.

Similar Intracellular Peptide Profile of TAP1/beta 2 Microglobulin Double-Knockout Mice and C57BL/6 Wild-Type Mice as Revealed by Peptidomic Analysis

Cells produce and use peptides in distinctive ways. In the present report, using isotope labeling plus semi-quantitative mass spectrometry, we evaluated the intracellular peptide profile of TAP1/beta 2m(-/-) (transporter associated with antigen-processing 1/beta 2 microglobulin) double-knockout mice and compared it with that of C57BL/6 wild-type animals. Overall, 92 distinctive peptides were identified, and most were shown to have a similar concentration in both mouse strains. However, some peptides showed a modest increase or decrease (similar to 2-fold), whereas a glycine-rich peptide derived from the C-terminal of neurogranin (KGPGPGGPGGAGGARGGAGGGPSGD) showed a substantial increase (6-fold) in TAP1/beta 2m(-/-) mice. Thus, TAP1 and beta 2microglobulin have a small influence on the peptide profile of neuronal tissue, suggesting that the presence of peptides derived from intracellular proteins in neuronal tissue is not associated with antigens of the class I major histocompatibility complex. Therefore, it is possible that these intracellular peptides play a physiological role.

TNF-alpha Knockout Mice Have Increased Corpora Cavernosa Relaxation

Erectile dysfunction is considered an early clinical manifestation of vascular disease and an independent risk factor for cardiovascular events associated with endothelial dysfunction and increased levels of pro-inflammatory cytokines. Tumor necrosis factor-alpha (TNF-alpha), a pro-inflammatory cytokine, suppresses endothelial nitric oxide synthase (eNOS) expression. Considering that nitric oxide (NO) is of critical importance in penile erection, we hypothesized that blockade of TNF-alpha actions would increase cavernosal smooth muscle relaxation. In vitro organ bath studies were used to measure cavernosal reactivity in wild type and TNF-alpha knockout (TNF-alpha KO) mice and NOS expression was evaluated by western blot. In addition, spontaneous erections (in vivo) were evaluated by videomonitoring the animals (30 minutes). Collagen and elastin expression were evaluated by Masson trichrome and Verhoff-van Gieson stain reaction, respectively. Corpora cavernosa from TNF-alpha KO mice exhibited increased NO-dependent relaxation, which was associated with increased eNOS and neuronal NOS (nNOS) cavernosal expression. Cavernosal strips from TNF-alpha KO mice displayed increased endothelium-dependent (97.4 +/- 5.3 vs. Control: 76.3 +/- 6.3, %) and nonadrenergic-noncholinergic (93.3 +/- 3.0 vs. Control: 67.5 +/- 16.0; 16 Hz) relaxation compared to control animals. These responses were associated with increased protein expression of eNOS and nNOS (P < 0.05). Sympathetic-mediated (0.69 +/- 0.16 vs. Control: 1.22 +/- 0.22; 16 Hz) as well as phenylephrine-induced contractile responses (1.6 +/- 0.1 vs. Control: 2.5 +/- 0.1, mN) were attenuated in cavernosal strips from TNF-alpha KO mice. Additionally, corpora cavernosa from TNF-alpha KO mice displayed increased collagen and elastin expression. In vivo experiments demonstrated that TNF-alpha KO mice display increased number of spontaneous erections. Corpora cavernosa from TNF-alpha KO mice display alterations that favor penile tumescence, indicating that TNF-alpha plays a detrimental role in erectile function. A key role for TNF-alpha in mediating endothelial dysfunction in ED is markedly relevant since we now have access to anti-TNF-alpha therapies. Carneiro FS, Sturgis LC, Giachini FRC, Carneiro ZN, Lima VV, Wynne BM, Martin SS, Brands MW, Tostes RC, and Webb RC. TNF-alpha knockout mice have increased corpora cavernosa relaxation. J Sex Med 2009;6:115-125.