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Intravascular injections of fluorescent or biotinylated tomato lectin were tested to study labeling of vascular elements in laboratory mice. Injections of Lycopersicon esculentum agglutinin (tomato lectin) (50–100 µg/100 µl) were made intravascularly, through the tail vein, through a cannula implanted in the jugular vein, or directly into the left ventricle of the heart. Tissues cut for thin 10- to 12-µm cryostat sections, or thick 50- to 100-µm vibratome sections, were examined using fluorescence microscopy. Tissue labeled by biotinylated lectin was examined by bright field microscopy or electron microscopy after tissue processing for biotin. Intravascular injections of tomato lectin led to labeling of vascular structures in a variety of tissues, including brain, kidney, liver, intestine, spleen, skin, skeletal and cardiac muscle, and experimental tumors. Analyses of fluorescence in serum indicated the lectin was cleared from circulating blood within 2 min. Capillary labeling was apparent in tissues collected from animals within 1 min of intravascular injections, remained robust for about 1 h, and then declined markedly until difficult to detect 12 h after injection. Light microscopic images suggest the lectin bound to the endothelial cells that form capillaries and endothelial cells that line some larger vessels. Electron microscopic studies confirmed the labeling of luminal surfaces of endothelial cells. Vascular labeling by tomato lectin is compatible with a variety of other morphological labeling techniques, including histochemistry and immunocytochemistry, and thus appears to be a sensitive and useful method to reveal vascular patterns in relationship to other aspects of parenchymal development, structure, and function.

Despite the discovery of heterotrimeric αβγ G proteins ∼25 years ago, their selective perturbation by cell-permeable inhibitors remains a fundamental challenge. Here we report that the plant-derived depsipeptide FR900359 (FR) is ideally suited to this task. Using a multifaceted approach we systematically characterize FR as a selective inhibitor of Gq/11/14 over all other mammalian Gα isoforms and elaborate its molecular mechanism of action. We also use FR to investigate whether inhibition of Gq proteins is an effective post-receptor strategy to target oncogenic signalling, using melanoma as a model system. FR suppresses many of the hallmark features that are central to the malignancy of melanoma cells, thereby providing new opportunities for therapeutic intervention. Just as pertussis toxin is used extensively to probe and inhibit the signalling of Gi/o proteins, we anticipate that FR will at least be its equivalent for investigating the biological relevance of Gq. Pertussis toxin is used extensively for perturbing Gαi/o pathways in the study of physiology and disease, but an equivalent inhibitor of Gαq signalling is not currently available to the research community. Here the authors characterize FR900359 as a specific Gq inhibitor and demonstrate its utility to dissect GPCR signalling and its potential to inhibit melanoma cells.

Estrogen withdrawal in menopausal women leads to hot flushes, a syndrome characterized by the episodic activation of heat dissipation effectors. Despite the extraordinary number of individuals affected, the etiology of flushes remains an enigma. Because menopause is accompanied by marked alterations in hypothalamic kisspeptin/neurokinin B/dynorphin (KNDy) neurons, we hypothesized that these neurons could contribute to the generation of flushes. To determine if KNDy neurons participate in the regulation of body temperature, we evaluated the thermoregulatory effects of ablating KNDy neurons by injecting a selective toxin for neurokinin-3 expressing neurons [NK ₃-saporin (SAP)] into the rat arcuate nucleus. Remarkably, KNDy neuron ablation consistently reduced tail-skin temperature (T SKIN), indicating that KNDy neurons facilitate cutaneous vasodilatation, an important heat dissipation effector. Moreover, KNDy ablation blocked the reduction of T SKIN by 17β-estradiol (E ₂), which occurred in the environmental chamber during the light phase, but did not affect the E ₂ suppression of T SKIN during the dark phase. At the high ambient temperature of 33 °C, the average core temperature (T CORE) of ovariectomized (OVX) control rats was significantly elevated, and this value was reduced by E ₂ replacement. In contrast, the average T CORE of OVX, KNDy-ablated rats was lower than OVX control rats at 33 °C, and not altered by E ₂ replacement. These data provide unique evidence that KNDy neurons promote cutaneous vasodilatation and participate in the E ₂ modulation of body temperature. Because cutaneous vasodilatation is a cardinal sign of a hot flush, these results support the hypothesis that KNDy neurons could play a role in the generation of flushes.

Although clinically apparent metastasis is associated with late stages of cancer development, micro-metastatic dissemination may be an early event. However, the fate of these early disseminated tumor cells (DTC) remains elusive. We show that despite their capacity to disseminate into secondary organs, 4T1 tumor models develop overt metastasis while EMT6-tumor bearing mice clear DTCs shed from primary tumors as well as those introduced by intravenous (IV) injection. Following the surgical resection of primary EMT6 tumors, mice do not develop detectable metastasis and reject IV-injected tumor cells. In contrast, these cells readily grow and metastasize in immuno-deficient athymic or Rag2 −/− mice, an effect mimicked by CD8 + T-cell depletion in immunocompetent mice. Furthermore, recombinant G-CSF or adoptive transfer of granulocytic-MDSCs isolated from 4T1 tumor-bearing mice, induce metastasis by suppressing CD8 + T-cells in EMT6-primed mice. Our studies support the concept of immune surveillance providing molecular insights into the immune mechanisms during tumor progression. Dissemination of tumor cells from the primary site is an early event. Here, the authors show that the early disseminated tumor cells are actively cleared by the host cytotoxic T lymphocytes induced by the primary tumor and that infiltration of granulocytic myeloid-derived suppressor cells counteracts such immune protection and allow metastasis development.

Background The American Heart Association has recommended tail-cuff blood pressure measurement for high-throughput experimental designs, including mutagenesis screens and genetic crosses. However, some tail-cuff methods show good agreement with radiotelemetry and others do not, indicating that each tail-cuff method requires independent validation. Methods We validated the volume–pressure recording (VPR) tail-cuff method by comparison to simultaneous radiotelemetry measurements. Results Bland–Altman analysis of 560 cycles from 26 independent measurement sessions showed good agreement between VPR and radiotelemetry measurements, with tail-cuff measurements being 0.25 mm Hg lower than telemetry measurements on average. However, the VPR method was less accurate, compared to radiotelemetry, at extreme high and low (i.e., <110 or >180 mm Hg) systolic blood pressures (SBPs). Conclusions We conclude that the VPR tail-cuff method provides accurate blood pressure measurements over the physiological range of blood pressure in mice.

Thyroid hormone is a major regulator of thermogenesis, acting both in peripheral organs and on central autonomic pathways. Mice heterozygous for a point mutation in thyroid hormone receptor α1 display increased thermogenesis as a consequence of high sympathetic brown fat stimulation. Surprisingly, despite the hypermetabolism, their body temperature is not elevated. Here we show, using isolated tail arteries, that defective thyroid hormone receptor α1 signaling impairs acetylcholine-mediated vascular relaxation as well as phenylephrine-induced vasoconstriction. Using infrared thermography on conscious animals, we demonstrate that these defects severely interfere with appropriate peripheral heat conservation and dissipation, which in turn leads to compensatory alterations in brown fat activity. Consequently, when the vasoconstrictive defect in mice heterozygous for a point mutation in thyroid hormone receptor α1 was reversed with the selective α1-adrenergic agonist midodrine, the inappropriate heat loss over their tail surface was reduced, normalizing brown fat activity and energy expenditure. Our analyses demonstrate that thyroid hormone plays a key role in vascular heat conservation and dissipation processes, adding a unique aspect to its well-documented functions in thermoregulation. The data thus facilitate understanding of temperature hypersensitivity in patients with thyroid disorders. Moreover, the previously unrecognized connection between cardiovascular regulation and metabolic activity revealed in this study challenges the interpretation of several experimental paradigms and questions some of the currently derived hypotheses on the role of thyroid hormone in thermogenesis.

This review first considers how mammalian body temperature regulation evolved, and how the brain’s responses to thermoregulatory challenges are likely to be organised differently from the way an engineer would design them. This is because thermoregulatory effector mechanisms would have evolved one at a time, with each being superimposed on pre-existing mechanisms. There may be no functional need for the final ensemble of control loops to be coordinated by neural cross-connections: appropriate thermal thresholds would solve the problem sufficiently. Investigations first into thermoregulatory behaviours and later into unconscious thermoregulatory mechanisms (autonomic and shivering) have led investigators to the realisation that multiple control loops exist in the brain, with each effector system apparently regulated by its own central temperature sensors. This theme is developed with reference to data on four temperature-regulated neural outflows that have been studied on anaesthetized rats under standard conditions in the authors’ laboratory. Direct comparisons were made between the behaviour of sympathetic nerves supplying the tail vasculature, vessels in the proximal hairy skin, interscapular brown adipose tissue (BAT) and fusimotor fibres to hind limb muscle. All four outflows were activated by cooling the skin, and all were silenced by neuronal inhibition in the medullary raphé. Their thermal thresholds were quite different, however, as were their relative responsiveness to core temperature. This was ranked as: tail > back skin > BAT > fusimotor. These and other data indicate that the four thermoeffector outflows are driven by separate neural pathways, each regulated by independent brain temperature sensors.

It has been reported that, in the spontaneously hypertensive rat (SHR) model of hypertension, different components of the G-protein/adenylate cyclase (AC)/Calcium-activated potassium channel of high conductance (BK) channel signaling pathway are altered differently. In the upstream part of the pathway (G-protein/AC), a comparatively low efficacy has been established, whereas downstream BK currents seem to be increased. Thus, the overall performance of this signaling pathway in SHR is elusive. For a better understanding, we focused on one aspect, the direct targeting of the BK channel by the G-protein/AC pathway and tested the hypothesis that the comparatively low AC pathway efficacy in SHR results in a reduced agonist-induced stimulation of BK currents. This hypothesis was investigated using freshly isolated smooth muscle cells from WKY and SHR rat tail artery and the patch-clamp technique. It was observed that: (1) single BK channels have similar current–voltage relationships, voltage-dependence and calcium sensitivity; (2) BK currents in cells with a strong buffering of the BK channel activator calcium have similar current–voltage relationships; (3) the iloprost-induced concentration-dependent increase of the BK current is larger in WKY compared to SHR; (4) the effects of activators of the PKA pathway, the catalytic subunit of PKA and the potent and selective cAMP-analogue Sp-5,6-DCl-cBIMPS on BK currents are similar. Thus, our data suggest that the lower iloprost-induced stimulation of the BK current in freshly isolated rat tail artery smooth muscle cells from SHR compared with WKY is due to the lower efficacy of upstream elements of the G-Protein/AC/BK channel pathway.

Microvascular dysfunction is a major cause of impaired wound healing seen in diabetic patients. Therefore, reestablishment of structural and functional microvasculature could be beneficial to promote wound healing in these patients. Angiopoietin-1 (Ang1) is a specific growth factor functioning to generate a stable and functional vasculature through the Tie2 and Tie1 receptors. Here we determined the effectiveness of cartilage oligomeric matrix protein (COMP)-Ang1, a soluble, stable, and potent form of Ang1, on promotion of healing in cutaneous wounds of diabetic mice. An excisional full-thickness wound was made in the dorsal side of the tail of diabetic (db/db) mice, and mice were then treated systemically with adenovirus (Ade) encoding COMP-Ang1 or with control virus encoding β-gal (Ade-β-gal) or treated topically with recombinant COMP-Ang1 protein or BSA. Time course observations revealed that mice treated with Ade-COMP-Ang1 or COMP-Ang1 protein showed accelerated wound closure and epidermal and dermal regeneration, enhanced angiogenesis and lymphangiogenesis, and higher blood flow in the wound region compared with mice treated with control virus or BSA. COMP-Ang1 promotion of wound closure and angiogenesis was not dependent on endothelial nitric oxide synthase or inducible nitric oxide synthase alone. Taken together, these findings indicate that COMP-Ang1 can promote wound healing in diabetes through enhanced angiogenesis, lymphangiogenesis, and blood flow.

This study aimed to investigate the impact of administration routes in establishing the Adriamycin (ADR)‐induced chronic kidney disease (CKD) model. Using BALB/c mice, we compared the effects of conventional tail‐vein injection (TV10, 10 mg/kg) to those of retro‐orbital sinus (orbital vein) injection (OV10, 10 mg/kg; OV8, 8 mg/kg). The results indicated that the OV10 group exhibited CKD pathology similar to the TV10 group, with both groups demonstrating significantly higher urinary albumin/creatinine ratio (p < 0.05), tubular injury (p < 0.05), and degree of renal fibrosis (p < 0.05) than the OV8 group. No significant differences were observed between the OV10 and TV10 groups in urinary albumin/creatinine ratio, tubular injury, and degree of renal fibrosis. These findings demonstrated that retro‐orbital administration of 10 mg/kg ADR induces comparable effects to conventional tail‐vein administration. This technique's technical simplicity may improve experimental efficiency, reproducibility, and animal welfare in CKD research. In conclusion, this study validates the utility of retro‐orbital injection in CKD model establishment, demonstrating its potential to standardize and improve the reliability of future CKD research protocols. This study compared the effects of conventional tail‐vein injection and retro‐orbital sinus injection in establishing an Adriamycin (ADR)‐induced chronic kidney disease (CKD) model in BALB/c mice, revealing that 10 mg/kg ADR administered through either route produced similar CKD pathology. The results suggest that retro‐orbital injection is a technically simpler and equally effective method, potentially enhancing experimental efficiency, reproducibility, and animal welfare in CKD research.