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Objective: The morphology of lesions in mouse models of osteoarthritis (OA) has not been comprehensively characterized, in part because current histological assessments of OA focus primarily on articular cartilage (AC). In the present study, sections of murine stifle joints with naturally occurring (aged animals) and surgically induced (destabilized medial meniscus, DMM) OA were examined using a newly developed histological grading scheme that includes quantitative measurements and semiquantitative grades to evaluate multiple joint tissues.Design: The data collected was analyzed using Principal Components Analysis (PCA); factor scores for each joint were generated. Individual parameters and factor scores were compared between surgical groups and among age groups. For comparison, the original Mankin Histological-Histochemical Grading System (HHGS) also was applied.Results: Overall, lesions were most severe in the medial tibial plateaus. Significant changes in AC and neighboring bone were identified in surgically induced models and in naturally occurring disease. Mean factor scores provided a comprehensive evaluation of joint changes. An important new finding was that chondrocyte cell death within the AC was a commonly identified lesion and its extent significantly increased with age. While the Mankin HHGS detected significant overall differences in OA severity between surgical groups, it was not sensitive in detecting age-related differences, nor did it provide information regarding changes in individual tissues.Conclusion: These results demonstrate the utility of this newly developed murine OA grading scheme in identifying lesions in AC and in other joint tissues. Surgically induced changes were similar to those occurring naturally with aging. [ABSTRACT FROM AUTHOR]

The burden of chronic soft tissue inflammation and neuropathic pain on individuals and society is substantial. This study was conducted to evaluate the H-wave device--an innovative form of treatment for chronic pain and inflammation--in patients with persistent pain associated with injuries or conditions affecting the upper or lower extremities or the back. Patients with at least moderate pain despite conventional therapy were included in a systematic survey after they had been given 2 to 6 wk of treatment with the H-wave device. Measures of improvement involved the proportion of patients with diminished medication requirements, improved function, or pain relief greater than 25%. More than 60% of patients with pain in the lower extremities, upper extremities, or back experienced pain relief exceeding 25%. The proportion of patients whose function improved and who were able to perform a new activity was consistently greater than 50% across the 3 anatomic subgroups. More than 40% of patients in each group were able to reduce or completely eliminate the use of pain medications. These benefits of treatment were independent of the type of pain therapy administered previously. In each anatomic subgroup, the proportion of patients who reported improvement on more than 1 of the 3 endpoints was significantly higher than the expected response to placebo therapy (P<.001). Results suggest that the H-wave device provided important benefits to patients with chronic soft tissue inflammation and neuropathic pain. [ABSTRACT FROM AUTHOR]

Background: The melanocortin 4 antagonist TCMCB07 is safe and effective in reversing cachexia caused by sepsis or cancer in rodents. The safety and pharmacokinetics of TCMCB07 are demonstrated in healthy beagle dogs., Hypothesis/objectives: The objectives of this study were to investigate the safety, peak plasma concentrations, and potential for efficacy of TCMCB07 in pet dogs with naturally occurring cachexia over a 4-week time period., Animals: Fourteen dogs with cachexia of any underlying cause, except cancer of the oral cavity or gastrointestinal tract, were eligible for enrollment with informed client consent., Methods: This study was a prospective, 1-armed open-label trial. Physical examination, complete blood count, chemistry panel, and owner-assessed quality of life surveys were checked at weeks 1, 2, and 4. Due to potential for bradycardia and hypotension, Holter monitoring and blood pressure evaluations were scheduled at pre-enrollment and week 4., Results: Fourteen dogs completed the trial. Significant changes detected included increased mean body weight (18.6-19.5 kg, P < .02), increased body condition score (median Tufts 5-point thin dog scale score P < .004 and WSAVA muscle condition score P < .02) and increased mean blood urea nitrogen (21.79-30.43 mg dL -1 , P < .004). On quality of life surveys, pet owners perceived their dog appeared to be panting less (P < .002) and that the general health improved (P < .03). Four dogs had a change in coat pigmentation. The peak plasma concentration of TCMCB07 in cachectic dogs was similar to that in healthy beagle dogs., Conclusions and Clinical Importance: TCMCB07 was safe and has potential efficacy in pet dogs with cachexia., (© 2023 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals LLC on behalf of American College of Veterinary Internal Medicine.)

Objective: Meniscus injury and the hypoxia-inducible factor (HIF) pathway are independently linked to osteoarthritis pathogenesis, but the role of the meniscus HIF pathway remains unclear. We sought to identify and evaluate HIF pathway response in normal and osteoarthritic meniscus and to examine the effects of Epas1 (HIF-2α) insufficiency in mice on early osteoarthritis development., Methods: Normal and osteoarthritic human meniscus specimens were obtained and used for immunohistochemical evaluation and cell culture studies for the HIF pathway. Meniscus cells were treated with pro-inflammatory stimuli, including interleukins (IL)-1β, IL-6, transforming growth factor (TGF)-α, and fibronectin fragments (FnF). Target genes were also evaluated with HIF-1α and HIF-2α (Epas1) overexpression and knockdown. Wild-type ( n = 36) and Epas1 +/- ( n = 30) heterozygous mice underwent destabilization of the medial meniscus (DMM) surgery and were evaluated at 2 and 4 weeks postoperatively for osteoarthritis development using histology., Results: HIF-1α and HIF-2α immunostaining and gene expression did not differ between normal and osteoarthritic meniscus. While pro-inflammatory stimulation significantly increased both catabolic and anabolic gene expression in the meniscus, HIF-1α and Epas1 expression levels were not significantly altered. Epas1 overexpression significantly increased Col2a1 expression. Both wild-type and Epas1 +/- mice developed osteoarthritis following DMM surgery. There were no significant differences between genotypes at either time point., Conclusion: The HIF pathway is likely not responsible for osteoarthritic changes in the human meniscus. Additionally, Epas1 insufficiency does not protect against osteoarthritis development in the mouse at early time points after DMM surgery. The HIF pathway may be more important for protection against catabolic stress.

Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) is upregulated in congestive heart failure (CHF), contributing to electrical, structural, and functional remodeling. CaMKII inhibition is known to improve CHF, but its direct cardiac effects in CHF remain unclear. We hypothesized that CaMKII inhibition improves cardiomyocyte function, [Ca 2+ ] i regulation, and β -adrenergic reserve, thus improving advanced CHF. In a 16-week study, we compared plasma neurohormonal levels and left ventricular (LV)- and myocyte-functional and calcium transient ([Ca 2+ ] iT ) responses in male Sprague-Dawley rats (10/group) with CHF induced by isoproterenol (170 mg/kg sq for 2 days). In rats with CHF, we studied the effects of the CaMKII inhibitor KN-93 or its inactive analog KN-92 ( n = 4) (70 µg/kg per day, mini-pump) for 4 weeks. Compared with controls, isoproterenol-treated rats had severe CHF with 5-fold-increased plasma norepinephrine and about 50% decreases in ejection fraction (EF) and LV contractility [slope of LV end-systolic pressure-LV end-systolic volume relation (E ES )] but increased time constant of LV relaxation ( τ ). They also showed significantly reduced myocyte contraction [maximum rate of myocyte shortening (dL/dt max )], relaxation (dL/dt max ), and [Ca 2+ ] iT Isoproterenol superfusion caused significantly fewer increases in dL/dt max and [Ca 2+ ] iT KN-93 treatment prevented plasma norepinephrine elevation, with increased basal and acute isoproterenol-stimulated increases in EF and E ES and decreased τ in CHF. KN-93 treatment preserved normal myocyte contraction, relaxation, [Ca 2+ ] iT , and β -adrenergic reserve, whereas KN-92 treatment failed to improve LV and myocyte function, and plasma norepinephrine remained high in CHF. Thus, chronic CaMKII inhibition prevented CHF-induced activation of the sympathetic nervous system, restoring normal LV and cardiomyocyte basal and β -adrenergic-stimulated contraction, relaxation, and [Ca 2+ ] iT , thereby playing a rescue role in advanced CHF. SIGNIFICANCE STATEMENT: We investigated the therapeutic efficacy of late initiation of chronic Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) inhibition on progression of advanced congestive heart failure (CHF). Chronic CaMKII inhibition prevented CHF-induced activation of the sympathetic nervous system and restored normal intrinsic cardiomyocyte basal and β -adrenergic receptor-stimulated relaxation, contraction, and [Ca 2+ ] i regulation, leading to reversal of CHF progression. These data provide new evidence that CaMKII inhibition is able and sufficient to rescue a failing heart, and thus cardiac CaMKII inhibition is a promising target for improving CHF treatment., Competing Interests: No conflicts of interest, financial or otherwise, are declared by the authors., (Copyright © 2021 by The American Society for Pharmacology and Experimental Therapeutics.)

The melanocortin-4 receptor (MC4R) antagonistic peptide TCMCB07 was developed for the treatment of cachexia. The objectives of this study were to examine pharmacokinetics and safety of TCMCB07 administered subcutaneously to healthy dogs. Dogs were treated with high- (2.25 mg kg -1 ) (n = 5) and low-dose TCMCB07 (0.75 mg kg -1 ) (n = 5) once daily for 28 days with a 14-day washout period between groups. Histamine levels, complete blood count, chemistry panel, blood pressure, 24-hour Holter recording, and pharmacokinetic parameters were monitored in the high-dose group. Physical examination changes were limited to weight gain and darkening of the coat color. There was no elevation of plasma histamine within 24 hours of injection but there was a significant elevation of plasma histamine across time. An approximately doubled eosinophil count and an approximately 25% increase, and then 25% decrease back to pre-treatment plasma phosphorous were also found, although both remained within the reference interval. Serial blood pressure and 24-hour Holter monitors revealed no clinically relevant changes. A difference was found in the AUC between dosing groups and a significant effect of dose, time, and interaction was noted for V d . Low-dose TCMCB07 had a C max of 2.1 ug ml -1 at day 28, compared to high-dose TCMCB07 which had a C max 3.6 ug ml -1 at day 28. Once-daily subcutaneous administration of TCMCB07 was well-tolerated for up to 28 days in dogs when administered at doses one and three times (0.75 mg kg -1 and 2.25 mg kg -1 ) the predicted therapeutic dose and pharmacokinetic parameters are described. SIGNIFICANCE STATEMENT: Melanocortin-4 receptor (MC4R) antagonistic peptide TCMCB07 is safe at both low and high doses in dogs. Therapy was tolerated well as determined by physical examination, clinical pathology, and cardiovascular parameters; darkening of the coat was noted with treatment and resolved with discontinuation. Pharmacokinetics are described and further study in the naturally occurring canine model is warranted., (© 2021 The Authors. Pharmacology Research & Perspectives published by British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics and John Wiley & Sons Ltd.)

Cachexia, a devastating wasting syndrome characterized by severe weight loss with specific losses of muscle and adipose tissue, is driven by reduced food intake, increased energy expenditure, excess catabolism, and inflammation. Cachexia is associated with poor prognosis and high mortality and frequently occurs in patients with cancer, chronic kidney disease, infection, and many other illnesses. There is no effective treatment for this condition. Hypothalamic melanocortins have a potent and long-lasting inhibitory effect on feeding and anabolism, and pathophysiological processes increase melanocortin signaling tone, leading to anorexia, metabolic changes, and eventual cachexia. We used 3 rat models of anorexia and cachexia (LPS, methylcholanthrene sarcoma, and 5/6 subtotal nephrectomy) to evaluate efficacy of TCMCB07, a synthetic antagonist of the melanocortin-4 receptor. Our data show that peripheral treatment using TCMCB07 with intraperitoneal, subcutaneous, and oral administration increased food intake and body weight and preserved fat mass and lean mass during cachexia and LPS-induced anorexia. Furthermore, administration of TCMCB07 diminished hypothalamic inflammatory gene expression in cancer cachexia. These results suggest that peripheral TCMCB07 treatment effectively inhibits central melanocortin signaling and therefore stimulates appetite and enhances anabolism, indicating that TCMCB07 is a promising drug candidate for treating cachexia.

., (Copyright © 2019 National Association of Pediatric Nurse Practitioners. Published by Elsevier Inc. All rights reserved.)

Objectives: To examine nurses' experiences of prognosis-related communication (PRC) with parents of children with cancer., Sample & Setting: Cross-sectional, correlational study in the pediatric oncology setting involving 316 members of the Association of Pediatric Hematology/Oncology Nurses., Methods & Variables: Online survey regarding individual nurse factors, PRC, interprofessional collaboration, moral distress, and perceived quality of care., Results: Nurses strongly agreed that prognostic disclosure is critical for decision making, but they are challenged in determining their role. Nurses with more years of experience and training in PRC, those working in an outpatient setting, and those with higher levels of nurse-physician collaboration reported more positive experiences with PRC. Positive experiences with PRC and collaboration were significantly associated with higher nurse-perceived quality of care and reduced nurse moral distress., Implications for Nursing: Nurses should work to be active participants in the process of PRC by collaborating with physician colleagues. When nurses sense that prognostic discussions have been absent or unclear, they should feel confident in approaching physician colleagues to ensure parent understanding and satisfaction with communication.

This study evaluated the potential impact of an online spiritual care educational program on pediatric nurses' attitudes toward and knowledge of spiritual care and their competence to provide spiritual care to children with cancer at the end of life. It was hypothesized that the intervention would increase nurses' positive attitudes toward and knowledge of spiritual care and increase nurses' level of perceived spiritual care competence. A positive correlation was expected between change in nurses' perceived attitudes toward and knowledge of spiritual care and change in nurses' perceived spiritual care competence. A prospective, longitudinal design was employed, and analyses included one-way repeated-measures analysis of variance, linear regression, and partial correlation. Statistically significant differences were found in nurses' attitudes toward and knowledge of spiritual care and nurses' perceived spiritual care competence. There was a positive relationship between change scores in nurses' attitudes toward and knowledge of spiritual care and nurses' spiritual care competence. Online spiritual care educational programs may exert a lasting impact on nurses' attitudes toward and knowledge of spiritual care and their competence to provide spiritual care to children with cancer at the end of life. Additional studies are required to evaluate the direct effects of educational interventions patient outcomes.

Background: Altered nitric oxide synthase (NOS) has been implicated in the pathophysiology of heart failure (HF). Recent evidence links hypothyroidism to the pathology of HF. However, the precise mechanisms are incompletely understood. The alterations and functional effects of cardiac NOS in hypothyroidism are unknown. We tested the hypothesis that hypothyroidism increases cardiomyocyte inducible NOS (iNOS) expression, which plays an important role in hypothyroidism-induced depression of cardiomyocyte contractile properties, [Ca(2+)]i transient ([Ca(2+)]iT), and β-adrenergic hyporesponsiveness., Methods and Results: We simultaneously evaluated LV functional performance and compared myocyte three NOS, β-adrenergic receptors (AR) and SERCA2a expressions and assessed cardiomyocyte contractile and [Ca(2+)]iT responses to β-AR stimulation with and without pretreatment of iNOS inhibitor (1400 W, 10(-5)mol/L) in 26 controls and 26 rats with hypothyroidism induced by methimazole (~30 mg/kg/day for 8 weeks in the drinking water). Compared with controls, in hypothyroidism, total serum T3 and T4 were significantly reduced followed by significantly decreased LV contractility (EES) with increased LV time constant of relaxation. These LV abnormalities were accompanied by concomitant significant decreases in myocyte contraction (dL/dtmax), relaxation (dR/dtmax), and [Ca(2+)]iT. In hypothyroidism, isoproterenol (10(-8)M) produced significantly smaller increases in dL/dtmax, dR/dtmax and [Ca(2+)]iT. These changes were associated with decreased β1-AR and SERCA2a, but significantly increased iNOS. Moreover, only in hypothyroidism, pretreatment with iNOS inhibitor significantly improved basal and isoproterenol-stimulated myocyte contraction, relaxation and [Ca(2+)]iT., Conclusions: Hypothyroidism produces intrinsic defects of LV myocyte force-generating capacity and relaxation with β-AR desensitization. Up-regulation of cardiomyocyte iNOS may promote progressive cardiac dysfunction in hypothyroidism., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Background: Radiation-induced fibrosis (RIF) of musculoskeletal tissue is a common complication of radiation therapy for extremity soft-tissue sarcoma, with no standardized strategy for prevention and treatment. Angiotensin-(1-7) (Ang-[1-7]), a well-tolerated endogenous heptapeptide hormone with antitumor and antifibrotic properties, was tested as a radioprotectant for RIF and stiffening of irradiated muscles., Methods: Male CD-1 mice were randomized to one of three treatment groups: control, simulated sarcoma radiation therapy to the gastrocnemius and soleus muscles, or radiation therapy along with continuous Ang-(1-7) delivery initiated three days before radiation therapy. The biologically equivalent dose of radiation (∼100.3 Gy) absorbed by normal musculature during the course of radiation therapy for extremity sarcoma was delivered by means of four dose fractions of 7.3 Gy over two weeks. Fibrosis (n = 5 per group) and mechanical properties (n = 4 to 6 per group) of the muscles were measured at six weeks and four months after radiation therapy, and the intramuscular concentration of the profibrotic cytokines transforming growth factor-beta (TGF-β) and connective tissue growth factor (CTGF) (n = 8 to 10 per group) were measured at six weeks., Results: Interstitial (p < 0.01) and perivascular (p < 0.05) fibrosis increased significantly in the muscles treated with radiation therapy alone versus the nonirradiated controls at both six weeks (interstitial, +89%; perivascular, +112%) and four months (interstitial, +154%; perivascular, +88%). The muscles treated with radiation alone also exhibited increased tension (p < 0.01) versus nonirradiated controls at both six weeks (+779%) and four months (+1761%) when placed under 5% strain, and at four months (+1390%; p < 0.001) under 10% strain. At four months, muscle stiffness had increased in the mice treated with radiation therapy alone (+90%; p = 0.002) compared with nonirradiated controls. TGF-β production was also greater in this group at six weeks (+37%; p = 0.06) versus control. Ang-(1-7) administration prevented RIF and stiffening, with no differences observed for any other outcome between those receiving radiation therapy with Ang-(1-7) and the nonirradiated controls. Likewise, Ang-(1-7) mitigated the increase in TGF-β and CTGF concentration from radiation therapy., Conclusions: Ang-(1-7) attenuated RIF, stiffening, and production of profibrotic cytokines that were elevated in mouse skeletal muscles after simulated radiation therapy for extremity sarcoma., Clinical Relevance: Ang-(1-7) may serve as a potential therapy for the prevention of RIF in patients who require radiation therapy as adjuvant treatment for soft-tissue sarcoma., (Copyright © 2016 by The Journal of Bone and Joint Surgery, Incorporated.)

Absorbable collagen sponges (ACS) are used clinically as carriers of recombinant human bone morphogenetic protein 2 (rhBMP-2) to promote bone regeneration. ACS exhibit ectopic bone growth due to delivery of supraphysiological levels of rhBMP-2, which is particularly problematic in craniofacial bone injuries for both functional and esthetic reasons. We hypothesized that hydrogels from the reduced form of keratin proteins (kerateine) would serve as a suitable alternative to ACS carriers of rhBMP-2. The rationale for this hypothesis is that keratin biomaterials degrade slowly in vivo, have modifiable material properties, and have demonstrated capacity to deliver therapeutic agents. We investigated kerateine hydrogels and freeze-dried scaffolds as rhBMP-2 carriers in a critically-sized rat mandibular defect model. ACS, kerateine hydrogels, and kerateine scaffolds loaded with rhBMP-2 achieved bridging in animals by 8 weeks as indicated by micro-computed tomography. Kerateine scaffolds achieved statistically increased bone mineral density compared to ACS and kerateine hydrogels, with levels reaching those of native bone. Importantly, both kerateine hydrogels and kerateine scaffolds had significantly less ectopic bone growth than ACS sponges at both 8 and 16 weeks post-operatively. These studies demonstrate the suitability of keratins as rhBMP-2 carriers due to equal regenerative capacity with reduced ectopic growth compared to ACS., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The ability to integrate theory with practice is integral to a student's success. A common reason for attrition from a nurse anesthesia program is clinical issues. To document clinical competence, students are evaluated using various tools. For use of a clinical evaluation tool as possible evidence for a student's dismissal, an important psychometric property to ensure is instrument validity. Clinical evaluation instruments of nurse anesthesia programs are not standardized among programs, which suggests a lack of instrument validity. The lack of established validity of the instruments used to evaluate students' clinical progress brings into question their ability to detect a student who is truly in jeopardy of attrition. Given this possibility, clinical instrument validity warrants research to be fair to students and improve attrition rates based on valid data. This ex post facto study evaluated a 17-item clinical instrument tool to demonstrate the need for validity of clinical evaluation tools. It also compared clinical scores with scores on the National Certification Examination.

Death after severe hemorrhage remains an important cause of mortality in people under 50 years of age. Keratin resuscitation fluid (KRF) is a novel resuscitation solution made from keratin protein that may restore cardiovascular stability. This postulate was tested in rats that were exsanguinated to 40% of their blood volume. Test groups received either low or high volume resuscitation with either KRF or lactated Ringer's solution. KRF low volume was more effective than LR in recovering cardiac function, blood pressure and blood chemistry. Furthermore, in contrast to LR-treated rats, KRF-treated rats exhibited vital signs that resembled normal controls at 1-week.

Intranasal administration has been widely used to investigate the effects of the neuropeptides vasopressin and oxytocin on human behaviour and neurological disorders, although exactly what happens when these neuropeptides are administered intranasally is far from clear. In particular, it is not clear whether a physiological significant amount of peptide enters the brain to account for the observed effects. In the present study, we investigated whether the intranasal administration of vasopressin and oxytocin to rats induces the expression of the immediate-early gene product Fos in brain areas that are sensitive to centrally-administered peptide, whether it alters neuronal activity in the way that centrally-administered peptide does, and whether it affects behaviour in the ways that are expected from studies of centrally-administered peptide. We found that, whereas i.c.v. injection of very low doses of vasopressin or oxytocin increased Fos expression in several distinct brain regions, intranasal administration of large doses of the peptides had no significant effect. By contrast to the effects of vasopressin applied topically to the main olfactory bulb, we saw no changes in the electrical activity of olfactory bulb mitral cells after intranasal vasopressin administration. In addition, vasopressin given intranasally had no significant effects on social recognition or short-term recognition memory. Finally, intranasal infusions of vasopressin had no significant effects on the parameters monitored on the elevated plus maze, a rodent model of anxiety. Our data obtained in rats suggest that, after intranasal administration, significant amounts of vasopressin and oxytocin do not reach areas in the brain at levels sufficient to change immediate early gene expression, neural activity or behaviour in the ways described for central administration of the peptides., (© 2013 British Society for Neuroendocrinology.)

Botulinum Neurotoxin A (BoNT-A) injections have been used for the treatment of muscle contractures and spasticity. This study assessed the influence of (BoNT-A) injections on passive biomechanical properties of the muscle-tendon unit. Mousegastrocnemius muscle (GC) was injected with BoNT-A (n = 18) or normal saline (n = 18) and passive, non-destructive, in vivo load relaxation experimentation was performed to examine how the muscle-tendon unit behaves after chemical denervation with BoNT-A. Injection of BoNT-A impaired passive muscle recovery (15% vs. 35% recovery to pre-stretching baseline, p < 0.05) and decreased GC stiffness (0.531 ± 0.061 N/mm vs. 0.780 ± 0.037 N/mm, p < 0.05) compared to saline controls. The successful use of BoNT-A injections as an adjunct to physical therapy may be in part attributed to the disruption of the stretch reflex; thereby modulating in vivo passive muscle properties. However, it is also possible that BoNT-A injection may alter the structure of skeletal muscle; thus modulating the in vivo passive biomechanical properties of the muscle-tendon unit.

Objective: To determine whether the ratio of dietary calcium and oxalate consumption at mealtime affects gastrointestinal oxalate absorption and urinary oxalate excretion., Methods: A study was conducted with 10 non-stone-forming adults placed on controlled diets with daily calcium and oxalate contents of 1000 and 750 mg, respectively. Subjects consumed a balanced calcium/oxalate ratio diet for 1 week, observed a minimum 1-week washout period, and subsequently consumed an imbalanced calcium/oxalate ratio diet for one week. Urine specimens were collected on the last 4 days of each diet. Outcome measures included urinary creatinine, calcium, and oxalate as well as the Tiselius index for assessing urinary calcium oxalate supersaturation., Results: Total daily calcium excretion, oxalate excretion, and Tiselius index were similar between balanced and imbalanced dietary phases. There were significant differences in calcium excretion (mg/g creatinine) between balanced and imbalanced diets in the 1-6 PM (83.1 vs 110.2, P <.04), 6-11 PM (71.3 vs 107.2, P <.02), and 11 PM-8 AM collections (55.0 vs 41.8, P <.02). There was significantly higher oxalate excretion on the balanced diet in the 1-6 pm time period (28.1 vs 16.7, P <.01). There were no differences in the Tiselius index in these collections., Conclusion: These results demonstrate that the sequence of ingesting relatively large amounts of oxalate does not significantly affect calcium oxalate stone risk if the recommended daily quantity of dietary calcium is consumed., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Purpose: Botulinum neurotoxin-A (BoNTA) is used to treat several disorders, including Raynaud phenomenon. Recent investigations cite toxin-induced increases in blood flow, but no mechanism for BoNTA's actions is proposed. This study hypothesized that local application of BoNTA causes arteriolar vasodilation through sympathetic blockade and results in increased blood flow., Methods: Microvascular effects of BoNTA were assessed using a rat cremaster preparation. Cremaster microvascular diameters were measured in the muscle before and after treatment with the muscle paralytic agent gallamine triethiodide. Preparations were then treated with one of the following: BoNTA (4, 6, or 10 units), BoNTA dilution vehicle, or denatured BoNTA. Arteriolar diameters were measured repeatedly over the observation period. Additional preparations were treated with either tetrodotoxin or prazosin and rauwolscine before BoNTA to confirm that the observed vasodilatory responses were the result of sympathetic neural inhibition., Results: The BoNTA application resulted in a significant dose-dependent vasodilation (13% to 15%) of observed cremaster arterioles. Control treatments did not cause vasodilation. Both tetrodotoxin and prazosin/rauwolscine treatments elicited similar vasodilatory effects, with no additional vasodilation elicited by BoNTA. Addition of sodium nitroprusside following BoNTA elicited further vasodilation. In addition, systemic arterial pressure was unaffected by the local administration of BoNTA., Conclusions: Local application of BoNTA results in arteriolar dilation that yields an approximate 69% increase in blood flow, without changing systemic arterial pressure. A BoNTA-mediated vasodilation through sympathetic blockade is a likely mechanism to explain the increase in blood flow reported after treatment with the toxin., Clinical Relevance: The ability of BoNTA to inhibit sympathetic nervous input reduces vasoconstriction, which is the most likely mechanism for improvement seen in Raynaud phenomenon patients following BoNTA injection., (Copyright © 2012 American Society for Surgery of the Hand. Published by Elsevier Inc. All rights reserved.)

Hydroxyproline (Hyp) metabolism is a key source of glyoxylate production in the body and may be a major contributor to excessive oxalate production in the primary hyperoxalurias where glyoxylate metabolism is impaired. Important gaps in our knowledge include identification of the tissues with the capacity to degrade Hyp and the development of model systems to study this metabolism and how to suppress it. The expression of mRNA for enzymes in the pathway was examined in 15 different human tissues. Expression of the complete pathway was identified in liver, kidney, pancreas, and small intestine. HepG2 cells also expressed these mRNAs and enzymes and were shown to metabolize Hyp in the culture medium to glycolate, glycine, and oxalate. [(18)O]- and [(13)C(5)]Hyp were synthesized and evaluated for their use with in vitro and in vivo models. [(18)O]Hyp was not suitable because of an apparent tautomerism of [(18)O]glyoxylate between enol and hydrated forms, which resulted in a loss of isotope. [(13)C(5)]Hyp, however, was metabolized to [(13)C(2)]glycolate, [(13)C(2)]glycine, and [(13)C(2)]oxalate in vitro in HepG2 cells and in vivo in mice infused with [(13)C(5)]Hyp. These model systems should be valuable tools for exploring various aspects of Hyp metabolism and will be useful in determining whether blocking Hyp catabolism is an effective therapy in the treatment of primary hyperoxaluria.

Objective: To better understand the contribution of age to the development of osteoarthritis (OA)., Methods: Surgical destabilization of the medial meniscus (DMM) was used to model OA in 12-week-old and 12-month-old male C57BL/6 mice. OA severity was evaluated histologically. RNA used for microarray and real-time polymerase chain reaction analysis was isolated from joint tissue collected from the medial side of the joint, including cartilage, meniscus, subchondral bone, and the joint capsule with synovium. Computational analysis was used to identify patterns of gene expression, and immunohistochemistry was used to evaluate tissue distribution of selected proteins., Results: OA was more severe in older mice than in young mice. Only 55 genes showed a similar expression with DMM-induced OA in the 2 age groups, while 493 genes showed differential expression, the majority having increased expression in older mice. Functional categories for similarly expressed genes included extracellular matrix- and cell adhesion-related genes; differentially expressed genes included those related to muscle structure and development and immune response genes. Comparison of expression in sham-operated control joints revealed an age-related decrease in matrix gene expression and an increase in immune and defense response gene expression. Interleukin-33 was present in multiple joint tissue cells, while CCL21 was more localized to chondrocytes and meniscal cells. Periostin was found in the extracellular matrix of cartilage and meniscus., Conclusion: Age affects both the basal pattern of gene expression in joint tissues and the response to surgically induced OA. Examining tissue from the joint beyond only cartilage revealed novel genes and proteins that would be important to consider in OA., (Copyright © 2012 by the American College of Rheumatology.)

Effects of repeated H-Wave® device stimulation (HWDS) on blood flow and angiogenesis in the rat hind limb were studied. The hypothesis tested was that HWDS acutely increases hind limb blood flow, and that repeated HWDS would elicit angiogenesis. Animals were HWDS-conditioned (``Conditioned'') or sham-stimulated (``Sham'') (n = 5/group) daily for 3 weeks. The contralateral limb in both groups served as the control. Each animal was injected with bromodeoxyuridine (BrDU). After 3 weeks, rats were anesthetized and iliac artery blood flow was measured bilaterally before, during, and after acute HWDS. HWDS of the Conditioned limbs elicited a 247% increase in blood flow above resting conditions compared to a 200% increase in control legs. Sham animals did not demonstrate between-leg differences in flow. Hindlimb musculature staining for BrDU revealed angiogenesis in Conditioned versus Sham groups. Flow changes accompanying HWDS corroborated earlier microvascular findings demonstrating a significant striated muscle arteriolar dilation with HWDS.

Botulinum neurotoxin-A (BoNTA) is a potent neurotoxin used to alter muscle tone to manage spasticity and to provide tendon bioprotection; however, the appropriate dose and injection volume to administer is not defined. Male mice (n = 120) received BoNTA injections into one gastrocnemius with either a constant volume (10 µl) with a variable dose (1, 3, 6 U/kg) or a constant dose (3 U/kg) in a variable volume (2.5, 5, 10, 20, 30 µl). Electromyographic (EMG) examination, muscle force generation (MFG), and wet muscle mass were measured in the ipsilateral and contralateral limbs at 1, 2, 4, or 12 weeks post-injection. MFG and EMG responses decreased to approximately 40% of contralateral after a 1 U/kg injection and 0% of contralateral by 3 and 6 U/kg injection at 1 week after injection. Neuromuscular blockade was greatest with a 10 µl injection volume. MFG, EMG examination, and wet muscle mass reached contralateral values 12 weeks after injection for all injection doses and volumes tested. Effective injection doses and volumes were identified for producing full and partial neuromuscular blockade in the mouse gastrocnemius. These findings have important clinical implications in the intramuscular administration of BoNTA to manage muscle tone., (Copyright © 2011 Orthopaedic Research Society.)

Objective: Accurate histological assessment of osteoarthritis (OA) is critical in studies evaluating the effects of interventions on disease severity. The purpose of the present study was to develop a histological grading scheme that comprehensively and quantitatively assesses changes in multiple tissues that are associated with OA of the stifle joint in mice., Design: Two representative midcoronal sections from 158 stifle joints, including naturally occurring and surgically induced OA, were stained with H&E and Safranin-O stains. All slides were evaluated to characterize the changes present. A grading scheme that includes both measurements and semiquantitative scores was developed, and principal components analysis (PCA) was applied to the resulting data from the medial tibial plateaus. A subset of 30 tibial plateaus representing a wide range of severity was then evaluated by 4 observers. Reliability of the results was evaluated using intraclass correlation coefficients (ICCs) and area under the receiver operating characteristic (ROC) curve., Results: Five factors were retained by PCA, accounting for 74% of the total variance. Interobserver and intraobserver reproducibilities for evaluations of articular cartilage and subchondral bone were acceptable. The articular cartilage integrity and chondrocyte viability factor scores were able to distinguish severe OA from normal, minimal, mild, and moderate disease., Conclusion: This newly developed grading scheme and resulting factors characterize a range of joint changes in mouse stifle joints that are associated with OA. Overall, the newly developed scheme is reliable and reproducible, characterizes changes in multiple tissues, and provides comprehensive information regarding a specific site in the stifle joint.

Background: Surgical repair of large chronic rotator cuff tears can be technically demanding because it requires manipulation of a muscle-tendon unit that is scarred, retracted, and stiffer than normal, all of which contribute to increased tension at the repair site. The purpose of the present study was to characterize the in vivo rotator cuff muscle-tendon unit function after acute and chronic injury at surgically relevant preload tensions., Methods: Sixty-two Sprague-Dawley rats were divided into a healthy, uninjured (control) group (n = 22), an acute injury group (n = 20), and a chronic injury group (n = 20) and underwent in vivo muscle force testing and electromyographic testing of the supraspinatus muscle-tendon unit at various preload tensions., Results: Preload tension affected the maximum supraspinatus muscle contractile force in all groups (p < 0.05). At the peak tension required to repair an acute tear, there was a 28% to 30% reduction in maximum tetanic contraction amplitude in all groups (p < 0.05). At the peak tension required to repair a chronic tear, there was a 40% to 53% reduction in maximal tetanic contraction amplitude in all groups (p < 0.05). The uninjured (control) group showed increased muscle endurance (p < 0.05) in comparison with the acute injury and chronic injury groups at all preload tensions. The chronic injury group showed reduced compound motor action potential amplitude (p < 0.05)., Conclusions: Both the acute and chronic injury groups demonstrated functional impairment related to increasing preload tensions. Higher repair tensions, associated with the chronic injury setting, resulted in greater functional impairment. The present study also demonstrates an association between increased time from rotator cuff tendon injury and impaired in vivo rotator cuff muscle electromyographic findings.

As part of a 5-year study funded by the National Cancer Institute, all graduate nursing students, including student registered nurse anesthetists (SRNAs) participated in a 2-credit-hour course called Interdisciplinary Palliative Care. Medical and health science students also participated in the course, with more than 800 students completing the course to date. The sample consisted of 62 master's-level students enrolled in either the first or second year of the nurse anesthesia program. A pretest-posttest design was used to determine changes in palliative care knowledge and perceived effectiveness in palliative care skills. There was an overall improvement in knowledge and attitudes related to course content. Students reported that, through the development of new knowledge, they felt better able to care for and advocate for their patients. Further research is needed into the appropriate roles that Certified Registered Nurse Anesthetists (CRNAs) can play in palliative and end-of-life care.

Experiments in humans and rodents using oral doses of glycine and phenylalanine have suggested that the metabolism of these amino acids contributes to urinary oxalate excretion. To better define this contribution, we have examined the primed, constant infusion of [1-(13)C(1)] phenylalanine and [1,2-(13)C(2)] glycine in the postabsorptive state in healthy adults. Subjects were infused for 5 hours, hourly urines were collected, and blood was drawn every 30 minutes. Ion chromatography/mass spectrometry was used to measure [(13)C] enrichment in urinary oxalate, glycolate, and hippurate; and the enrichment of (13)C-amino acids in plasma samples was measured by gas chromatography/mass spectrometry. Following infusion with either 6 μmol/(kg h) [1-(13)C(1)] phenylalanine or 6 μmol/(kg h) [1,2-(13)C(2)] glycine, no isotopic glycolate or oxalate was detected in urine. Based on the limits of detection of our ion chromatography/mass spectroscopy method, these data indicate that less than 0.7% of the urinary oxalate could be derived from phenylalanine catabolism and less than 5% from glycine catabolism. Infusions with high levels of [1,2-(13)C(2)] glycine, 60 μmol/(kg h), increased mean plasma glycine by 29% and the whole-body flux of glycine by 72%. Under these conditions, glycine contributed 16.0% ± 1.6% and 16.6% ± 3.2% to urinary oxalate and glycolate excretion, respectively. Experiments using cultured hepatoma cells demonstrated that only at supraphysiological levels (>1 mmol/L) did glycine and phenylalanine metabolism increase oxalate synthesis. These data suggest that glycine and phenylalanine metabolism make only minor contributions to oxalate synthesis and urinary oxalate excretion., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Purpose: The chemical denervation that results from botulinum neurotoxin A (BoNT-A) causes a temporary, reversible paresis that can result in easier surgical manipulation of the muscle-tendon unit in the context of tendon rupture and repair. The purpose of the study was to determine whether BoNT-A injections can be used to temporarily and reversibly modulate active and passive skeletal muscle properties., Methods: Male CD1 mice weighing 40-50 g were divided into a 1-week postinjection group (n = 13: n = 5 saline and n = 8 BoNT-A) and a 2-week postinjection group (n = 17: n = 7 saline and n = 10 BoNT-A). The animals had in vivo muscle force testing and in vivo biomechanical evaluation., Results: There was a substantial decline in the maximal single twitch amplitude (p < .05) and tetanic amplitude (p < .05) at one week and at 2 weeks after BoNT-A injection, when compared to saline-injected controls. BoNT-A injection significantly reduced the peak passive properties of the muscle-tendon unit as a function of displacement at one week (p < .05). Specifically, the stiffness of the BoNT-A injected muscle-tendon unit was 0.417 N/mm compared to the control saline injected group, which was 0.634 N/mm, a 35% reduction in stiffness (p < .05)., Conclusions: Presurgical treatment with BoNT-A might improve the surgical manipulation of the muscle-tendon unit, thus improving surgical outcomes. The results implicate neural tone as a substantial contributor to the passive repair tension of the muscle-tendon unit. The modulation of neural tone through temporary, reversible paresis is a novel approach that might improve intraoperative and postoperative passive muscle properties, allowing for progressive rehabilitation while protecting the surgical repair site., (Copyright © 2011 American Society for Surgery of the Hand. Published by Elsevier Inc. All rights reserved.)

H-Wave electrical device stimulation (HWDS) is used clinically to expedite recovery from soft tissue injuries. We hypothesized that HWDS induces arteriolar dilation, a mechanism involved in the healing process. Acute effects of HWDS on striated muscle arteriolar diameters were studied. Arteriolar diameters were measured in the cremaster muscle of 57 male anesthetized rats using intravital microscopy before and after HWDS or sham stimulation (SS) at 1 or 2 Hz for periods of 30-60 min. In a separate cohort, the role of nitric oxide (NO) in the response to HWDS was assessed by blocking NO synthase using topical L-NAME at 10(-5) M. Maximal arteriolar responses to stimulation were compared to prestimulation diameters. HWDS both at 1 and 2 Hz resulted in significant arteriolar vasodilation (p < 0.05). The arterioles in SS animals demonstrated no changes in diameter. Similarly, microvascular diameters did not change with HWDS following blockade of NO production. Because of Poiseuille's Law, the significant arteriolar dilation induced by HWDS would translate into increases in blood flow of 26-62%. In addition, lack of arteriolar dilation following HWDS with blockade of NO production suggests that NO plays a role in the microvascular response to HWDS. These studies suggest that arteriolar vasodilation accompanying HWDS may result in increased perfusion, contributing to the observed therapeutic effects of HWDS., ((c) 2009 Orthopaedic Research Society.)

The current study determined the dose-response relationship for inhibition of muscle force of two commercially available botulinum neurotoxin type-A (BoNTA) preparations (Botox and Neuronox) in a murine model and characterized the time course of recovery from the toxin-induced muscle paralysis. The effect of freezing reconstituted toxin on toxin potency was also determined. The gastrocnemius muscles in male CD-1 mice were injected with either saline or BoNTA (0.3-3.0 U/kg), and muscle force generation was examined following stimulation of the tibial nerve (single twitch and 15-200 Hz tetany). Botox and Neuronox produced nearly equivalent decrements in muscle force (30%-90%) at 4 days after toxin injection. At 28 days after injection (1 U/kg), muscle force had recovered from the effects of both toxin preparations. Maintaining reconstituted toxin at -80 degrees C for up to 5 months did not result in significant loss of toxin activity. The results of this study suggest that Botox and Neuronox produce equivalent responses in a murine model, and, in contrast to other models, muscle recovery is rapid with doses of toxin that produce less than maximal decrements in muscle force., (Copyright 2007 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

Angiotensin-(1-7) [Ang-(1-7)] is an endogenous peptide of the renin-angiotensin system with vasodilator and antiproliferative properties. Our previous studies showed that Ang-(1-7) reduced serum-stimulated growth of human lung cancer cells in vitro through activation of a unique AT((1-7)) receptor. The current study investigates the effect of Ang-(1-7) on lung tumor growth in vivo, using a human lung tumor xenograft model. Athymic mice with tumors resulting from injection of A549 human lung cancer cells were treated for 28 days with either i.v. saline or Ang-(1-7), delivered by implanted osmotic mini-pumps. Treatment with Ang-(1-7) reduced tumor volume by 30% compared with the size before treatment; in contrast, tumor size in the saline-treated animals increased 2.5-fold. These results correlate with a reduction in the proliferation marker Ki67 in the Ang-(1-7)-infused tumors when compared with the saline-infused tumor tissues. Treatment with Ang-(1-7) significantly reduced cyclooxygenase-2 (COX-2) mRNA and protein in tumors of Ang-(1-7)-infused mice when compared with mice treated with saline as well as in the parent A549 human lung cancer cells in tissue culture. These results suggest that Ang-(1-7) may decrease COX-2 activity and proinflammatory prostaglandins to inhibit lung tumor growth. In contrast, the heptapeptide had no effect on COX-1 mRNA in xenograft tumors or A549 cells. Because Ang-(1-7), a peptide with antithrombotic properties, reduces growth through activation of a selective AT((1-7)) receptor, our results suggest that the heptapeptide represents a novel treatment for lung cancer by reducing COX-2.

The aim of this study was to compare the systemic hemodynamic effects of four commonly used anesthetic regimens in mice that were chronically instrumented for direct and continuous measurements of cardiac output (CO). Mice (CD-1, Swiss, and C57BL6 strains) were instrumented with a transit-time flow probe placed around the ascending aorta for CO measurement. An arterial catheter was inserted into the aorta 4 or 5 days later for blood pressure measurements. After full recovery, hemodynamic parameters including stroke volume, heart rate, CO, mean arterial pressure (MAP), and total peripheral resistance were measured with animals in the conscious state. General anesthesia was then induced in these mice using isoflurane (Iso), urethane, pentobarbital sodium, or ketamine-xylazine (K-X). The doses and routes of administration of these agents were given as required for general surgical procedures in these animals. Compared with the values obtained for animals in the conscious resting state, MAP and CO decreased during all anesthetic interventions, and hemodynamic effects were smallest for Iso (MAP, -24 +/- 3%; CO, -5 +/- 7%; n = 15 mice) and greatest for K-X (MAP, -51 +/- 6%; CO, -37 +/- 9%; n = 8 mice), respectively. The hemodynamic effects of K-X were fully antagonized by administration of the alpha(2)-receptor antagonist atipamezole (n = 8 mice). These results indicate that the anesthetic Iso has fewer systemic hemodynamic effects in mice than the nonvolatile anesthetics.

Glycyl-glutamine (Gly-Gln; beta-endorphin(30-31)) is an endogenous dipeptide that is synthesized through the posttranslational processing of beta-endorphin in brain stem regions that control respiration and autonomic function. This study tested the hypothesis that Gly-Gln administration to conscious rats will prevent the respiratory depression caused by morphine without affecting morphine antinociception. Rats were administered Gly-Gln (1-100 nmol) or saline (10 microl) intracerebroventricularly followed, 5 min later, by morphine (40 nmol icv). Arterial blood gases and pH were measured immediately before Gly-Gln and 30 min after morphine injection. Gly-Gln pretreatment inhibited morphine-induced hypercapnia, hypoxia, and acidosis significantly. The response was dose dependent and significant at Gly-Gln doses as low as 1 nmol. In contrast, Gly-Gln (1-300 nmol) had no effect on morphine-evoked antinociception in the paw withdrawal test. When given alone to otherwise untreated animals, Gly-Gln did not affect nociceptive latencies or blood gas values. These data indicate that Gly-Gln inhibits morphine-induced respiratory depression without compromising morphine antinociception.

Mice lacking AT(1A) receptors for ANG II have a defect in urinary concentration manifested by an inability to increase urinary osmolality to levels seen in controls after thirsting. This defect results in extreme serum hypertonicity during water deprivation. In the basal state, plasma vasopressin levels are similar in wild-type controls and Agtr1a -/- mice. Plasma vasopressin levels increase normally in the AT(1A) receptor-deficient mice after 24 h of water deprivation, suggesting that the defect in urine concentration is intrinsic to the kidney. Using magnetic resonance microscopy, we find that the absence of AT(1A) receptors is associated with a modest reduction in the distance from the kidney surface to the tip of the papilla. However, this structural abnormality seems to play little role in the urinary concentrating defect in Agtr1a -/- mice since the impairment is largely reproduced in wild-type mice by treatment with an AT(1)-receptor antagonist. These studies demonstrate a critical role for the AT(1A) receptor in maintaining inner medullary structures in the kidney and in regulating renal water excretion.

We previously demonstrated that the Ren-2 transgenic (TG) rat is sensitive to salt, showing a sodium-induced pressor response. The present studies determined the effect of central stimulation with hypertonic saline (HS) and angiotensin II (Ang II) on mean arterial pressure (MAP), heart rate (HR), and plasma vasopressin. HS (1 mol/L NaCl, 5 microL) or Ang II (100 ng, 5 microL) was injected into the lateral ventricle of conscious male TG and control rats. The pressor responses to HS and Ang were greater in TG than in control rats, increases of 42+/-4 and 41+/-4 mm Hg versus 25+/-3 and 18+/-2 mm Hg (HS and Ang II and TG and control rats, respectively). The TG rats also showed an increased vasopressin response to Ang II, peak levels of 14+/-3 versus 28+/-3 pg/mL (control versus TG rats). HS increased plasma vasopressin levels, although the group responses were not different. HR was not significantly altered by either stimulus. Results demonstrate an increased responsiveness to intraventricular HS and Ang II in Ren-2 transgenic rats, suggesting a relationship between the enhanced angiotensinergic drive and central cardiovascular and vasopressin responses.

Angiotensin (Ang) type 1a (AT1a) receptors are critical in the control of blood pressure and water balance. Experiments were performed to determine the influence of dehydration on brain Ang receptors and plasma vasopressin (VP) in mice lacking this receptor. Control or AT1a knockout (AT1aKO) male mice were give water ad libitum or deprived of water for 48 hours. Animals were anesthetized with halothane, blood samples were collected by heart puncture, and brains were processed for Ang-receptor autoradiography with 125I-sarthran (0.4 nmol/L). Dehydration produced an increase in AT1 receptors in the paraventricular nucleus (PVN) and anterior pituitary (AP) in control mice (PVN: 70+/-16 versus 146+/-10 fmol/mg protein; AP: 41+/-7 versus 86+/-15 fmol/mg protein). No changes were noted in the median preoptic nucleus. The majority of the brain receptors were of the AT1 subtype. There was little or no specific Ang binding in AT1aKO mice and no effect of dehydration. Plasma VP levels were elevated in the halothane-anesthetized animals (>200 pg/mL) with no significant effect of dehydration. A separate experiment was performed with decapitated mice anesthetized with pentobarbital. Dehydration increased plasma VP in control mice, from 3.3+/-0.6 to 13.3+/-4.7 pg/mL, whereas no change was noted in the AT1aKO mice, 5.1+/-0.3 versus 6.1+/-0.7 pg/mL (water versus dehydration). These results demonstrate a differential response to dehydration in mice lacking AT1a receptors. There was no evidence for AT1 receptors of any subtype in the brain regions examined and no effect of dehydration on VP secretion or brain Ang receptors.

We previously demonstrated that mRen-2 transgenic [Tg(+)] rats are sensitive to chronic high NaCl intake, showing increased arterial pressure and vasopressin (VP) secretion. In this study, we determined the effect of a chronic osmotic challenge, 4 days of drinking 2% NaCl, on direct arterial blood pressure, heart rate, fluid-electrolyte balance, circadian rhythm of mean arterial pressure (MAP), and changes in plasma VP and catecholamines. Under baseline conditions, male Tg(+) rats showed a significant shift in the peak in circadian MAP into the light portion of the day-night cycle. Substitution of 2% NaCl for drinking water caused a rapid increase in MAP, 20 +/- 5 mmHg in Tg(+) rats within 6 h. Whereas the amplitude of circadian MAP fluctuations increased in salt-loaded Tg(+) rats, there was no significant change in the circadian timing of peak MAP with salt loading. Tg(+) rats showed exaggerated osmotic-induced increases in plasma VP, norepinephrine (NE), and epinephrine (Epi) compared with Tg(-) rats. Plasma NE and Epi were increased two- and fourfold, respectively, in the hypertensive rats with no significant change in the Tg(-) rats. Intravenous administration of a VP antagonist did not alter arterial pressure in either Tg(+) or Tg(-) rats. Tg(+) and Tg(-) rats showed a positive sodium balance with no significant difference observed between the groups. Tg(+) rats showed a significant increase in salt consumption, plasma sodium, osmolality, and hematocrit, accompanied by a negative water balance. We conclude that Tg(+) rats are sensitive to acute and chronic osmotic stimuli in terms of blood pressure, fluid-electrolyte balance, and plasma VP and catecholamines. Whereas elevated plasma VP does not contribute to the hypertensive response, increased sympathetic drive may mediate the salt-induced blood pressure changes in this model.

The (mRen-2)27 transgenic rat (Tg+), a hypertensive model dependent on increased expression of the renin angiotensin system, was used to explore the role of angiotensin AT2 receptors in the control of cardiovascular and renal excretory function. Experiments tested the effect of blockade of AT2 receptors on basal blood pressure and the pressor, renal excretory, and vasopressin (VP) responses to intravenous hypertonic saline (HS). Chronically catheterized male Tg+ and normotensive Sprague-Dawley rats (Tg-) were housed in metabolic cages. PD123319 (AT2 antagonist) or 0.9% NaCl was given by intravenous bolus (3 mg/kg) followed by infusion (50 microg/kg/ min). Blockade of AT2 receptors both in Tg+ and Tg- rats produced no change in basal mean arterial pressure (MAP). The pressor response to intravenous HS (10% NaCl; 325 microL/100 g body weight) was significantly greater in Tg+ than in Tg- rats. PD123319 did not affect the peak rise in MAP but extended the time course of the response only in Tg+ rats. MAP was increased 39+/-4 and 36+/-3 mm Hg in Tg+ rats with and without the antagonist as compared to 20+/-2 and 24+/-2 mm Hg in Tg- rats. In the antagonist-treated Tg+ rats, MAP remained elevated for 60 min as compared to 5 min for Tg+ control or Tg- control or antagonist-treated rats. Hypertonic saline caused similar increases in plasma Na, VP, and in the natriuretic and diuretic responses in both Tg+ and Tg- rats, with no effect of antagonist treatment. These results demonstrate that Tg+ rats are sensitive to the effects of peripheral osmotic stimulation showing an increased pressor response, not attributed to greater secretion of VP or diminished natriuresis. These data also suggest that angiotensin AT2 receptors play a depressor role in the sodium-induced pressor response in this model.

Evidence indicates that the hypothalamic paraventricular nucleus (PVN) and oxytocin (OT) neurons in particular play a role in the physiological response to stress. Microdialysis (MD) experiments were performed to determine whether OT is released into the PVN during shaker stress. Male rats were prepared with venous catheters and PVN guide cannulae. OT and vasopressin (VP) release into PVN and peripheral blood were measured under basal conditions and during and after shaker stress (10 min at 110 cycles/min). Stress produced a specific increase in PVN and plasma OT. Dialysate OT levels were 0.3+/-0.1, 2.8+/-1.2 and 1.3+/-0.6 pg/sample (control, stress and recovery, respectively). Plasma OT was significantly increased during stress (3.7+/-1.2 vs. 11.7+/-2.3 pg/ml, basal vs. stress, respectively). When MD probes were located outside the PVN, there was no increase in OT release, demonstrating site specificity. Stress produced no change in VP levels, either in dialysate or plasma. These results show that OT, but not VP, is released into the PVN and peripheral blood in response to shaker stress. The data raise the possibility that local release of OT into the PVN plays a role in the neuroendocrine stress cascade., (Copyright 1998 Elsevier Science B.V.)

Sinoaortic baroreceptor denervation (SAD) results in increased osmotically induced secretion of vasopressin (VP) and oxytocin (OT) and increased cardiovascular responses to many centrally acting pressor agents. Studies were conducted to determine whether SAD increases the cardiovascular and endocrine responses to direct and peripheral osmotic stimulation of the supraoptic nucleus (SON). SON microdialysis was performed in urethane-anesthetized male rats with measurement of dialysate peptides, mean arterial pressure (MAP) and heart rate. Experiment 1 tested the effect of direct stimulation of the SON with hypertonic NaCl in SAD, sham-operated (control) and intake-matched (matched) rats. Osmotically induced VP release into the SON was significantly greater in SAD than in control or matched groups. VP release peaked at 36 +/- 13 and 15 +/- 7 pg in SAD and controls, respectively, with no increase observed in the matched group. Plasma VP was significantly elevated after SON osmotic stimulation with no differences observed among the groups. The pressor response to osmotic stimulation was greater in SAD (29 +/- 4 mm Hg) than in control (20 +/- 3 mm Hg) and matched animals (15 +/- 3 mm Hg). Experiment 2 tested the effect of intraperitoneal injection of hypertonic NaCl on SON VP and OT release. SAD rats showed an increased central VP response to peripheral osmotic stimulation, a 64-fold increase in SAD as compared to a 4-fold one in controls. Central OT release was not significantly altered (peak of 22 +/- 6 vs. 11 +/- 4 pg, SAD vs. control). A direct SON osmotic challenge given 3.5 h after the intraperitoneal test confirmed an increased VP responsiveness in the SAD group. Plasma VP and OT were significantly increased after intraperitoneal hypertonic saline with no difference observed between groups. The MAP response to intraperitoneal hypertonic saline was greater in the SAD group with an elevation of 37 +/- 4 versus 18 +/- 3 mm Hg observed in SAD versus control subjects. These results demonstrate that baroreceptor denervation produces a state of heightened osmotic sensitivity for VP neurons, with evidence for increased central VP release to both direct and peripheral hypertonic NaCl stimulation.

Baroreceptor input plays a critical role in body fluid balance and the endocrine response to NaCl consumption [25]. Experiments were performed to characterize the alterations in salt intake that are seen after baroreceptor denervation. Using chronically baroreceptor denervated (SAD) or control (CON) male Sprague-Dawley rats, we determined: 1) concentration-dependent consumption of NaCl, 2) time course of saline intake, 3) effect of food access on saline intake, 4) intake of sucrose vs. saline, and 5) water vs. saline intake using a choice paradigm. In protocols 1-4 the rats were given a single bottle containing saline or sucrose for a 2-h period during the early dark period. A comparison of the intake of varying concentrations of NaCl (0.3 to 2.0% NaCl, six concentrations) demonstrated that the SAD consumed significantly less NaCl than the CON (from 0.9 to 2% NaCl), Saline Intake in SAD was 14-56% of the CON (significant group, salt concentration and interaction effects). Regression analysis demonstrated that in the SAD there was an inverse relationship between concentration and the amount of NaCl consumed (p < 0.02), an effect not seen in the CON. There were also differences in the pattern of saline intake with the CON showing the highest consumption in the early dark period with a gradual decrease as compared to the SAD, which demonstrated a uniformly lower pattern of consumption. The reduction in intake in the SAD appeared to be specific for NaCl because there was no difference in water or sucrose intake. The deficit could not be attributed to alterations in food intake, nor was there any difference in the amount of water consumed after the saline challenge.

Central and systemic osmotic stimulation increase vasopressin (VP) release within the supraoptic nucleus (SON) and into the general circulation. We examined whether changes in water/electrolyte balance affect the neurosecretory responses to these stimuli. Urethane-anesthetized control, salt-loaded (2% NaCl for 2 days) or water-deprived (for 2 days) male rats were implanted with an arterial catheter and bilateral microdialysis probes into the SON. Plasma and SON VP levels were measured before and after acute osmotic stimuli were administered intraperitoneally (i.p.) and then directly into the SON. Water deprivation resulted in elevated basal intranuclear and plasma VP levels. Intraperitoneal hypertonic saline (HS) and direct osmotic stimulation of the SON increased VP release into the SON in both the control and water-deprived groups. Salt loading abolished the intranuclear VP response to both stimuli. Osmotically induced release of VP into plasma was not different between the three groups. These data demonstrate that salt loading, but not water deprivation, alters the central neurosecretory VP response to acute osmotic stimulation.

The effects of lesioning of the anteroventral third ventricle (AV3V) region on vasopressin (VP) release into the supraoptic nucleus (SON) and blood in response to central and systemic osmotic stimulation were determined. Microdialysis probes were implanted bilaterally within the SON of male urethan-anesthetized rats with sham or AV3V lesions. Osmotic stimuli were administered intraperitoneally (3.5 M NaCl, 600 microliters/100 g body wt) and then via the microdialysis probes (1 M NaCl-artificial cerebrospinal fluid). AV3V lesions attenuated the response to systemic osmotic stimulation. The lesioned rats showed no increase in intranuclear VP release and reduced plasma VP (increase of 42.6 +/- 8.4 vs. 78.0 +/- 16.4 pg/ml) and blood pressure responses (7.1 +/- 2.3 vs. 19.6 +/- 3.2 mmHg) to intraperitoneal NaCl. In contrast, the endocrine and cardiovascular responses to direct osmotic stimulation of the nucleus were as seen in previous studies and seemed to be unaffected by the lesion. These results show that lesion of the AV3V region interrupts neuronal inputs which trigger VP secretion from the posterior pituitary as well as release into the extracellular compartment of the SON.

We have previously demonstrated that mRen-2 transgenic [Tg(+)] rats show a salt-induced exacerbation of hypertension (Callaha, M., P. Li, C. M. Ferrario, D. Ganten, and M. Morris. Hypertension Dallas 27: 573-577, 1996). In this study, we examined the role of paraventricular (PVN) angiotensin type-1 (AT1) receptors in the salt sensitivity of this model. Male Tg(+) and Tg(-) rats were instrumented with PVN cannulas for intracerebral drug administration and carotid catheters for chronic cardiovascular monitoring. Substitution of 2% NaCl for drinking water for 4 days caused a significant elevation (23 mmHg) of mean arterial pressure (MAP) in Tg(+) rats but not in Tg(-) rats. PVN injection of AT1 receptor antisense oligodeoxynucleotides (ASODN), but not scrambled oligodeoxynucleotides (SCODN), produced a rapid decrease in MAP of 24 +/&#95; 8 mmHg in salt-treated Tg(+) rats. There was no effect of either AT1 ASODN or SCODN on MAP in salt-loaded Tg(-) rats or in Tg(+) rats consuming tap water. Salt loading significantly increased subfornical organ AT1 receptors in Tg(+) rats with no changes produced by ASODN or SCODN. In contrast, there was a 40% decrease in PVN AT1 receptors 20 h after direct PVN injection of AT1 ASODN injection, compared with SCODN in Tg(+) rats. We conclude that PVN AT1 receptors are critical in the expression of salt sensitivity in mRen-2 transgenic rats.

The (mREN-2)27 transgenic model of hypertension was developed to investigate the effect of genetic over activity of angiotensin II systems as a contributing factor in the development of arterial hypertension. In this model, transgene-positive rats demonstrate elevated renin-angiotensin system activity not only in the circulatory system but also in adrenal gland, reproductive organs, and brain. Since evidence indicates that angiotensin peptides and osmotic stimuli interact synergistically to produce exaggerated behavioral, endocrine, and cardiovascular effects, we examined the effect of salt consumption on arterial pressure, plasma vasopressin, and body fluid balance in male (mREN-2)27 transgene-positive and -negative rats. Four days of drinking 2% NaCl increased mean arterial pressure from 165 +/- 10 to 199 +/- 7 mm Hg in transgene-positive rats. In contrast, transgene-negative rats showed no change in arterial pressure (126 +/- 5 to 128 +/- 3 mm Hg). Plasma vasopressin levels were significantly elevated only in transgene-positive rats, whereas pituitary levels of vasopressin were significantly lower in transgene-positive rats compared with transgene-negative controls (18 +/- 3 and 118 +/- 14 ng, respectively). Although transgene-positive rats consumed significantly more 2% NaCl than did transgene-negative rats, during this period 24-hour sodium balance did not differ between the groups. Since fluid and electrolyte balance is similar between the two groups of rats, the data suggest that transgene-positive rats may be more sensitive to the effects of increased NaCl intake in terms of both endocrine and cardiovascular responses.

Tetrodotoxin (TTX) was used to (1) distinguish between axonal and dendritic/somatic release of vasopressin (VP) and oxytocin (OT) within the supraoptic nucleus (SON) and (2) to determine whether neuronal inputs trigger intranuclear peptide release in the response to osmotic stimulation. Microdialysis was used to administer TTX (10(-6) M or 10(-4)M) bilaterally into the SON with simultaneous monitoring of central and peripheral peptide release and mean arterial pressure in urethane-anesthetized male rats. Osmotic stimuli were given via the microdialysis probe (1 M NaCl-artificial CSF) or injected intraperitoneally (3.5 M NaCl; 600 mu l/100 g b.w.) SON perfusion with TTX did not alter basal intranuclear VP or OT release or the intranuclear peptide response to direct NaCl stimulation of the SON. However, TTX treatment abolished the effect of peripheral osmotic stimulation on central peptide release. Basal plasma peptide levels were significantly reduced by TTX, e.g. decreases of 94.8 and 75.8% for VP and OT, respectively. TTX also blocked the peripheral endocrine and cardiovascular responses to both modes of osmotic stimulation. The TTX insensitivity of directly stimulated intranuclear release suggests nonsynaptic peptide release from dendrites and/or cell bodies. The ability of TTX to abolish the central peptide response to systemic osmotic stimulation demonstrates that intranuclear release is a part of a cascade produced by osmotic activation of multisynaptic pathways.

Experiments were performed to evaluate the role of central oxytocin (OT) in the inhibition of salt intake produced by sinoartic denervation (SAD). The effect of OT antisense treatment on 24 h intake of 2% NaCl in SAD and sham-operated (SO) rats was determined. PVN injection of unmodified antisense oligodeoxynucleotides (ODNs) to OT mRNA decreased intake of 2% NaCl in SAD, but not SO rats. Salt consumption was 22 +/- 4 ml after the injection of control ODN as compared to 8 +/- 4 ml after the OT antisense injection (P < 0.05). SAD animals also demonstrated an increased plasma OT response to salt loading, an elevation from 3.2 +/- 0.7 to 6.9 +/- 0.8 pg/ml. In contrast, salt ingestion produced no significant change in plasma OT in the SO group. The increased endocrine response in the SADs occurred even though salt intake was lower in this group. There were no group differences in plasma electrolytes or posterior pituitary OT content. Results show that OT antisense specifically inhibits salt intake in the denervated rat, suggesting that the central oxytocinergic axis stimulates sodium drive in this experimental model.

To address the role of oxytocin in the control of cardiovascular reactivity, we examined the effect of central injection of oxytocin, vasopressin and mixed base antisense oligodeoxynucleotides on stress-induced cardiovascular and endocrine changes. Antisense oligomers were injected into the paraventricular nucleus (PVN), 4 h prior to the stress test. The oxytocin antisense abolished the tachycardia produced by 5 min of shaker stress. The blood pressure and plasma oxytocin responses were not different between the groups. PVN levels of OT were reduced in the oxytocin antisense-treated group while brain stem levels were increased. These results demonstrate the importance of a specific peptide system, the PVN/oxytocin axis, in stress-induced tachycardia. Further, the data illustrate the effectiveness of short-term treatment with antisense oligomers on physiological responses.

The effect of hypertonic NaCl consumption on vasopressin (VP) and oxytocin (OT) mRNA levels and plasma and pituitary peptides was evaluated in rats with sham or anterior ventral third ventricular (AV3V) lesions. Rats were given tap water or 2% NaCl for 4 days. Because the rats with lesions drank significantly less salt solution than the controls (78.8 +/- 17.4 vs. 205.5 +/- 37.8 ml/4 days), a second control group was included in which saline intake was matched to the lesioned group. AV3V rats showed a deficit in the peptide response to the osmotic stimulus. There was no increase in plasma VP or OT or decrease in posterior pituitary peptide content in the face of an extreme hypernatremia: plasma sodium of 180.1 +/- 4.2 meq/l. Evaluation of mRNA changes by means of in situ hybridization showed that animals with lesions responded to the salt challenge with increases in hypothalamic VP and OT mRNA levels. There were significant increases in paraventricular and supraoptic OT mRNA and paraventricular VP mRNA in the lesioned group. The salt-matched control group showed no changes in peptide mRNA levels. These results demonstrate that AV3V lesions produce an impairment of the salt-neuroendocrine reflex but a persistence of the peptide mRNA response. Differences in control mechanisms must account for this dissociation between peptide mRNA expression and peptide secretion.

Vasopressin (VP) and oxytocin (OT) are released within the hypothalamic nuclear region in response to direct microdialysis with hypertonic solutions. Experiments were performed to determine whether systemic osmotic stimulation causes changes in intranuclear peptide release within the supraoptic nucleus (SON). A hypertonic sodium chloride solution was injected intraperitoneally (i.p.) or intravenously (i.v.) and microdialysis techniques were used to simultaneously monitor central and peripheral peptide release in urethane anesthetized rats. Systemic osmotic stimuli elicited increases in intranuclear peptide release which were delayed and long-lasting, occurring over a 2.5 h period. In contrast, plasma peptide levels peaked at 30-min after the stimulus. The results demonstrate that increased plasma sodium elicits an increase in VP and OT release into the extracellular space of the hypothalamic SON. The different patterns of peptide release in plasma and brain point toward the possibility of independently regulated release into the different compartments.