Characterization of the anisotropic mechanical behaviour of colonic tissues: experimental activity and constitutive formulation
The aim was to investigate the biomechanical behaviour of colonic tissues by a coupled experimental and numerical approach. The wall of the colon is composed of different tissue layers. Within each layer, different fibre families are distributed according to specific spatial orientations, which lead to a strongly anisotropic configuration. Accounting for the complex histology of the tissues, mechanical tests must be planned and designed to evaluate the behaviour of the colonic wall in different directions. Uni-axial tensile tests were performed on tissue specimens from 15 fresh pig colons, accounting for six different loading directions (five specimens for each loading direction). The next step of the investigation was to define an appropriate constitutive framework and develop a procedure for identification of the constitutive parameters. A specific hyperelastic formulation was developed that accounted for the multilayered conformation of the colonic wall and the fibre-reinforced configuration of the tissues. The parameters were identified by inverse analyses of the mechanical tests. The comparison of model results with experimental data, together with the evaluation of satisfaction of material thermomechanics principles, confirmed the reliability of the analysis developed. This work forms the basis for more comprehensive activities that aim to provide computational tools for the interpretation of surgical procedures that involve the gastrointestinal tract, considering the specific biomedical devices adopted.
Cerebral hypoxaemia associated with rapid ascent to high altitude can be life threatening; yet, with proper acclimatization, cerebral function can be maintained well enough for humans to thrive. We investigated adjustments in global and regional cerebral oxygen delivery (DO2) as 21 healthy volunteers rapidly ascended and acclimatized to 5260 m. Ultrasound indices of cerebral blood flow in internal carotid and vertebral arteries were measured at sea level, upon arrival at 5260 m (ALT1; atmospheric pressure 409 mmHg) and after 16 days of acclimatization (ALT16). Cerebral DO2 was calculated as the product of arterial oxygen content and flow in each respective artery and summed to estimate global cerebral blood flow. Vascular resistances were calculated as the quotient of mean arterial pressure and respective flows. Global cerebral blood flow increased by ~70% upon arrival at ALT1 (P < 0.001) and returned to sea-level values at ALT16 as a result of changes in cerebral vascular resistance. A reciprocal pattern in arterial oxygen content maintained global cerebral DO2 throughout acclimatization, although DO2 to the posterior cerebral circulation was increased by ~25% at ALT1 (P = 0.032). We conclude that cerebral DO2 is well maintained upon acute exposure and acclimatization to hypoxia, particularly in the posterior and inferior regions of the brain associated with vital homeostatic functions. This tight regulation of cerebral DO2 was achieved through integrated adjustments in local vascular resistances to alter cerebral perfusion during both acute and chronic exposure to hypoxia.
Intermittent and continuous high-intensity exercise training induce similar acute but different chronic muscle adaptations
High-intensity interval training (HIIT) performed in an ‘all-out’ manner (e.g. repeated Wingate tests) is a time-efficient strategy to induce skeletal muscle remodelling towards a more oxidative phenotype. A fundamental question that remains unclear, however, is whether the intermittent or ‘pulsed’ nature of the stimulus is critical to the adaptive response. In study 1, we examined whether the activation of signalling cascades linked to mitochondrial biogenesis was dependent on the manner in which an acute high-intensity exercise stimulus was applied. Subjects performed either four 30 s Wingate tests interspersed with 4 min of rest (INT) or a bout of continuous exercise (CONT) that was matched for total work (67 ± 7 kJ) and which required ~4 min to complete as fast as possible. Both protocols elicited similar increases in markers of adenosine monophosphate-activated protein kinase (AMPK) and p38 mitogen-activated protein kinase activation, as well as Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) mRNA expression (main effects for time, P ≤ 0.05). In study 2, we determined whether 6 weeks of the CONT protocol (3 days per week) would increase skeletal muscle mitochondrial content to a similar extent to what we have previously reported after 6 weeks of INT. Despite similar acute signalling responses to the CONT and INT protocols, training with CONT did not increase the maximal activity or protein content of a range of mitochondrial markers. However, peak oxygen uptake was higher after CONT training (from 45.7 ± 5.4 to 48.3 ± 6.5 ml kg–1 min–1; P < 0.05) and 250 kJ time trial performance was improved (from 26:32 ± 4:48 to 23:55 ± 4:16 min:s; P < 0.001) in our recreationally active participants. We conclude that the intermittent nature of the stimulus is important for maximizing skeletal muscle adaptations to low-volume, all-out HIIT. Despite the lack of skeletal muscle mitochondrial adaptations, our data show that a training programme based on a brief bout of high-intensity exercise, which lasted <10 min per session including warm-up, and performed three times per week for 6 weeks, improved peak oxygen uptake in young healthy subjects.
Polycystic ovary syndrome (PCOS) is one of the most common endocrine disorders in women, with a prevalence of 8–12% during the reproductive years. In the present study, using prenatal exposure to a single dose of testosterone during the critical period of fetal development, we aimed to introduce an enhanced rat model that would exhibit both endocrine and ovarian disturbances similar to PCOS, while maintaining normal reproductive system morphology in adulthood. Ten pregnant rats were injected s.c. with 5 mg free testosterone on gestational day 20, while control rats received only solvent. The development and function of the reproductive system in female offspring were examined in adulthood. Prenatally androgenized offspring had irregular oestrous cycles compared with control animals, and their anogenital and anovaginal distances were increased compared with control rats (P < 0.001). No significant differences were observed in the lengths of the vagina and clitoris or the number of nipples between the two groups. Levels of testosterone and luteinizing hormone and the luteinizing hormone/follicle-stimulating hormone ratio were increased in prenatally androgenized offspring compared with control animals (P < 0.05). The numbers of preantral and antral follicles in the ovaries of prenatally androgenized offspring were also increased compared with control rats (P = 0.07 and P < 0.01, respectively). The number of corpora lutea was decreased in prenatally androgenized offspring compared with control rats. Cystic follicles were observed in the ovaries of prenatally androgenized offspring. Prenatal exposure to a single dose of testosterone during the critical period of fetal development could facilitate the development a functional rat model of PCOS in adulthood, with minimal morphological disorders in the reproductive system.
A plasminogen activator inhibitor type 1 mutant retards diabetic nephropathy in db/db mice by protecting podocytes
A mutant non-inhibiting plasminogen activator inhibitor type 1 (PAI-1), termed PAI-1R, which reduces endogenous PAI-1 activity, has been shown to inhibit albuminuria and reduce glomerulosclerosis in experimental diabetes. The mechanism of the reduction of albuminuria is unclear. This study sought to determine whether the administration of PAI-1R protected podocytes from injury directly, thereby reducing albuminuria in the db/db mouse, a model of type 2 diabetes. Untreated uninephrectomized db/db mice developed significant mesangial matrix expansion and albuminuria at week 22 of age, associated with segmental podocyte foot-process effacement, reduction of renal nephrin, podocin and zonula occludin-1 production and induction of renal desmin and B7-1 generation. In contrast, treatment with PAI-1R at 0.5 mg (kg body weight)–1 i.p., twice daily from week 20 to 22, reduced glomerular matrix accumulation, fibronectin and collagen production and albuminuria by 36, 62, 65 and 31%, respectively (P < 0.05), without affecting blood glucose level or body weight. Podocyte morphology and protein markers were also significantly attenuated by PAI-1R administration. Importantly, recombinant PAI-1 downregulated nephrin and zonula occludin-1 but increased desmin and B7-1 mRNA expression and protein production by podocytes in vitro, similar to the effects of transforming growth factor-β1. These observations provide evidence that PAI-1, in a manner similar to transforming growth factor-β1, directly induces podocyte injury, particularly in the setting of diabetes, where elevated PAI-1 may contribute to the progression of albuminuria. Reducing the increased PAI-1 activity by administration of PAI-1R, in fact, reduces podocyte injury, thereby reducing albuminuria. Therefore, PAI-1R provides an additional therapeutic effect in slowing the progression of diabetic nephropathy via the protection of podocytes.
Renal epithelial sodium channel is critical for blood pressure maintenance and sodium balance in the normal late pregnant rat
Normal pregnancy is a state marked by avid sodium retention and plasma volume expansion. Insufficient plasma volume expansion results in the compromised maternal state of intrauterine growth restriction, which afflicts ~5% of all human pregnancies. We have recently shown that renal epithelial sodium channel (ENaC) activity in vivo in the late pregnant (LP) rat is increased. To determine the importance of the renal versus extrarenal ENaC in sodium retention and blood pressure regulation during pregnancy, we have chronically blocked the ENaC pharmacologically with daily subcutaneous injections of benzamil and genetically using intrarenal transfection of αENaC short hairpin RNA. Compared with untreated LP control animals, LP rats treated with benzamil retain less sodium and have reduced mean arterial blood pressure. Furthermore, LP rats treated with benzamil had lower maternal body weight gain. Intrarenal transfection of αENaC short hairpin RNA versus scrambled small RNA successfully decreased renal αENaC mRNA expression in LP rats. Intrarenal transfection of αENaC short hairpin RNA reduced maternal sodium retention, body weight gain and pup weight. Redundant physiological systems that protect blood pressure and sodium homeostasis were unable to compensate for the loss of ENaC activity in the pregnant rat. These findings demonstrate that the renal ENaC is necessary for maintaining pregnancy-mediated sodium retention, volume expansion and blood pressure regulation.
Testosterone potentiates the hypoxic ventilatory response of adult male rats subjected to neonatal stress
Neonatal stress disrupts development of homeostatic systems. During adulthood, male rats subjected to neonatal maternal separation (NMS) are hypertensive and show a larger hypoxic ventilatory response (HVR), with greater respiratory instability during sleep. Neonatal stress also affects sex hormone secretion; hypoxia increases circulating testosterone of NMS (but not control) male rats. Given that these effects of NMS are not observed in females, we tested the hypothesis that testosterone elevation is necessary for the stress-related increase of the HVR in adult male rats. Pups subjected to NMS were placed in an incubator for 3 h per day from postnatal day 3 to 12. Control pups remained undisturbed. Rats were reared until adulthood, and the HVR was measured by plethysmography (fractional inspired O2 = 0.12, for 20 min). We used gonadectomy to evaluate the effects of reducing testosterone on the HVR. Gonadectomy had no effect on the HVR of control animals but reduced that of NMS animals below control levels. Immunohistochemistry was used to quantify androgen receptors in brainstem areas involved in the HVR. Androgen receptor expression was generally greater in NMS rats than in control rats; the most significant increase was noted in the caudal region of the nucleus tractus solitarii. We conclude that the abnormal regulation of testosterone is important in stress-related augmentation of the HVR. The greater number of androgen receptors within the brainstem may explain why NMS rats are more sensitive to testosterone withdrawal. Based on the similarities of the cardiorespiratory phenotype of NMS rats and patients suffering from sleep-disordered breathing, these results provide new insight into its pathophysiology, especially sex-based differences in its prevalence.
Differential modulation of sympathetic and respiratory activities by cholinergic mechanisms in the nucleus of the solitary tract in rats
The contribution of cholinergic mechanisms of the nucleus of the solitary tract (NTS) to cardiorespiratory control is not completely clear. In the present study, we investigated the involvement of the cholinergic mechanisms in the intermediate NTS (iNTS) and commissural NTS (cNTS) on the control of sympathetic (SNA) and phrenic nerve activity (PNA). Decorticated, arterially perfused in situ preparations of male juvenile rats (60–100 g) were used. Acetylcholine (10 mm, 60 nl) injected into the iNTS reduced SNA (–54 ± 4%, versus vehicle –5 ± 3%; P < 0.001) and PNA (–30 ± 4%, versus vehicle –5 ± 6%; P < 0.001), whereas injections of ACh into the cNTS increased PNA (30 ± 6%, versus vehicle 5 ± 3%; P < 0.001), without changing SNA. Pretreatment with mecamylamine (nicotinic antagonist; 5 mm) abolished all the effects of ACh injected into the iNTS or the cNTS, whereas atropine (muscarinic antagonist; 5 mm) reduced only the effects of ACh injected into the cNTS. Mecamylamine injected into the cNTS also reduced the tachypnoea in response to peripheral chemoreflex activation. The baroreflex was unaltered by injections of atropine or mecamylamine into the NTS. The results suggest that ACh and mainly nicotinic receptors in the NTS are involved in the modulation of SNA and PNA, with distinct functions between the iNTS and the cNTS. An involvement of the nicotinic receptors in the cNTS in the tachypnoea in response to peripheral chemoreflex activation is also suggested.
Reduced aerobic capacity, as measured by maximal oxygen uptake, is a hallmark in cardiovascular diseases and strongly predicts poor prognosis and higher mortality rates in heart failure patients. While exercise capacity is poorly correlated with cardiac function in this population, skeletal muscle abnormalities present a striking association with maximal oxygen uptake. This fact draws substantial attention to the clinical relevance of targeting skeletal myopathy in heart failure. Considering that skeletal muscle is highly responsive to aerobic exercise training, we addressed the benefits of aerobic exercise training to combat skeletal myopathy in heart failure, focusing on the mechanisms by which aerobic exercise training counteracts skeletal muscle atrophy.
What is the topic of this review?
Genetic modifiers act on many different physiological aspects of muscle disease. Understanding and identifying such modifiers is important because their discovery may help to predict the course of muscle disease and also indicate pathways to be exploited in designing new therapeutics.
What advances does it highlight?
Genetic modifiers have been identified that act primarily on limb skeletal muscles. Newer modifiers, where the responsible gene has yet to be identified, alter the course of cardiopulmonary dysfunction in muscular dystrophy. Distinct modifiers that act differentially on limb skeletal muscles versus heart and respiratory muscles reflect underlying physiological differences of these muscle groups.
Many single-gene disorders are associated with a range of symptoms that cannot be explained solely by the primary genetic mutation. Muscular dystrophy is a genetic disorder associated with variable outcomes that arise from both the primary genetic mutation and the contribution from environmental and genetic modifiers. Disruption of the dystrophin complex occurs in Duchenne muscular dystrophy and limb girdle muscular dystrophy, producing heart and muscle disease through a cellular injury process characterized by plasma membrane disruption and fibrosis. Multiple modifier loci have been mapped by using a mouse model of muscular dystrophy. These modifiers exert their effect often on specific muscle groups targeted by the muscular dystrophy process, possibly reflecting distinct pathophysiological processes among muscle groups. Genetic modifiers act on both cardiac and respiratory muscle parameters, suggesting genetic and physiological integration of cardiopulmonary function. Skeletal muscles of the limbs are modified by a locus on mouse chromosome 7. This region of chromosome 7 harbours an insertion/deletion polymorphism in Ltbp4, the gene encoding latent transforming growth factor β binding protein 4. LTBP4 exerts its effect in muscle disease by acting on plasma membrane stability and fibrosis, thereby linking instability of the sarcolemma directly to fibrosis. In the human muscle disease Duchenne muscular dystrophy, protein coding single-nucleotide polymorphisms in LTBP4 associate with prolonged ambulation, demonstrating that modifiers identified from mouse studies translate to human disease.
Autonomic, locomotor and cardiac abnormalities in a mouse model of muscular dystrophy: targeting the renin-angiotensin system
What is the topic of this review?
This symposium report summarizes autonomic, cardiac and skeletal muscle abnormalities in sarcoglycan--deficient mice (Sgcd–/–), a mouse model of limb girdle muscular dystrophy, with emphasis on the roles of autonomic dysregulation and activation of the renin–angiotensin system at a young age.
What advances does it highlight?
The contributions of the autonomic nervous system and the renin–angiotensin system to the pathogenesis of muscular dystrophy are highlighted. Results demonstrate that autonomic dysregulation precedes and predicts later development of cardiac dysfunction in Sgcd–/– mice and that treatment of young Sgcd–/– mice with the angiotensin type 1 receptor antagonist losartan or with angiotensin-(1–7) abrogates the autonomic dysregulation, attenuates skeletal muscle pathology and increases spontaneous locomotor activity.
Muscular dystrophies are a heterogeneous group of genetic muscle diseases characterized by muscle weakness and atrophy. Mutations in sarcoglycans and other subunits of the dystrophin–glycoprotein complex cause muscular dystrophy and dilated cardiomyopathy in animals and humans. Aberrant autonomic signalling is recognized in a variety of neuromuscular disorders. We hypothesized that activation of the renin–angiotensin system contributes to skeletal muscle and autonomic dysfunction in mice deficient in the sarcoglycan- (Sgcd) gene at a young age and that this early autonomic dysfunction contributes to the later development of left ventricular (LV) dysfunction and increased mortality. We demonstrated that young Sgcd–/– mice exhibit histopathological features of skeletal muscle dystrophy, decreased locomotor activity and severe autonomic dysregulation, but normal LV function. Autonomic regulation continued to deteriorate in Sgcd–/– mice with age and was accompanied by LV dysfunction and dilated cardiomyopathy at older ages. Autonomic dysregulation at a young age predicted later development of LV dysfunction and higher mortality in Sgcd–/– mice. Treatment of Sgcd–/– mice with the angiotensin type 1 receptor blocker losartan for 8–9 weeks, beginning at 3 weeks of age, decreased fibrosis and oxidative stress in skeletal muscle, increased locomotor activity and prevented autonomic dysfunction. Chronic infusion of the counter-regulatory peptide angiotensin-(1–7) resulted in similar protection. We conclude that activation of the renin–angiotensin system, at a young age, contributes to skeletal muscle and autonomic dysfunction in muscular dystrophy. We speculate that the latter is mediated via abnormal sensory nerve and/or cytokine signalling from dystrophic skeletal muscle to the brain and contributes to age-related LV dysfunction, dilated cardiomyopathy, arrhythmias and premature death. Therefore, correcting the early autonomic dysregulation and renin–angiotensin system activation may provide a novel therapeutic approach in muscular dystrophy.
Muscle disuse and starvation are often associated with a catabolic response leading to a dramatic loss of skeletal muscle mass. Hibernating animals represent a unique situation where muscle mass is maintained despite prolonged periods of immobilization and lack of nutrition. We analysed the molecular pathways upregulated during hibernation in an obligate hibernator, the 13-lined ground squirrel (Ictidomys tridecemlineatus). Although Akt has an established role in skeletal muscle maintenance, we found that activated Akt was decreased in skeletal muscle of hibernating squirrels. Another serine–threonine kinase, serum- and glucocorticoid-regulated kinase 1 (SGK1), was upregulated during hibernation and contributed to protection from loss of muscle mass via downregulation of proteolysis and autophagy and via an increase in protein synthesis. We extended our observations to non-hibernating animals and demonstrated that SGK1-null mice developed muscle atrophy. These mice displayed an exaggerated response to immobilization and starvation. Furthermore, SGK1 overexpression prevented immobilization-induced muscle atrophy. Taken together, our results identify SGK1 as a novel therapeutic target to combat skeletal muscle loss in acquired and inherited forms of muscle atrophy.
Role of the carotid body chemoreceptors in baroreflex control of blood pressure during hypoglycaemia in humans
Activation of the carotid body chemoreceptors with hypoxia alters baroreceptor-mediated responses. We aimed to examine whether this relationship can be translated to other chemoreceptor stimuli (i.e. hypoglycaemia) by testing the following hypotheses: (i) activation of the carotid body chemoreceptors with hypoglycaemia would reduce spontaneous cardiac baroreflex sensitivity (sCBRS) in healthy humans; and (ii) desensitization of the carotid chemoreceptors with hyperoxia would restore sCBRS to baseline levels during hypoglycaemia. Ten young healthy adults completed two 180 min hyperinsulinaemic [2 mU (kg fat-free mass)–1 min–1], hypoglycaemic (~3.2 μmol ml–1) clamps, separated by at least 1 week and randomized to normoxia (arterial partial pressure of O2, 122 ± 10 mmHg) or hyperoxia (arterial partial pressure of O2, 424 ± 123 mmHg; to blunt activation of the carotid body glomus cells). Changes in heart rate, blood pressure, plasma catecholamines, heart rate variability (HRV) and sCBRS were assessed. During hypoglycaemia, HRV and sCBRS were reduced (P < 0.05) and the baroreflex working range was shifted to higher heart rates. When hyperoxia was superimposed on hypoglycaemia, there was a greater reduction in blood pressure and a blunted rise in heart rate when compared with normoxic conditions (P < 0.05); however, there was no detectable effect of hyperoxia on sCBRS or HRV during hypoglycaemia (P > 0.05). In summary, hypoglycaemia-mediated changes in HRV and sCBRS cannot be attributed exclusively to the carotid chemoreceptors; however, the chemoreceptors appear to play a role in resetting the baroreflex working range during hypoglycaemia.
Angiotensin-converting enzyme (ACE and ACE2) imbalance correlates with the severity of cerulein-induced acute pancreatitis in mice
Angiotensin-converting enzyme (ACE) and its effector peptide angiotensin II (Ang II) have been implicated in the pathogenesis of pancreatitis. Angiotensin-converting enzyme 2 (ACE2) degrades Ang II to angiotensin-(1–7) [Ang-(1–7)] and has recently been described to have an antagonistic effect on ACE signalling. However, the specific underlying role of ACE2 in the pathogenesis of severe acute pancreatitis (SAP) is unclear. In the present study, the local imbalance of ACE and ACE2, as well as Ang II and Ang-(1–7) expression, was compared in wild-type (WT) and ACE2 knock-out (KO) or ACE2 transgenic (TG) mice subjected to cerulein-induced SAP. Serum amylase, tumour necrosis factor-α, interleukin (IL)-1β, IL-6 and IL-10 levels and histological morphometry were used to determine the severity of pancreatitis. In WT mice, pancreatic ACE and Ang II and serum Ang II expression increased (P < 0.05), while pancreatic ACE2 and Ang-(1–7) and serum Ang-(1–7) levels were also significantly elevated (P < 0.05) from 2 to 72 h after the onset of SAP. However, the ratio of pancreatic ACE2 to ACE expression was significantly reduced (from 1.46 ± 0.09 to 0.27 ± 0.05, P < 0.001) and paralleled the severity of pancreatitis. The Ace2 KO mice exhibited increased levels of tumour necrosis factor-α, IL-1β, IL-6, multifocal coagulative necrosis and inflammatory infiltrate, and lower levels of serum IL-10 and pancreatic Ang-(1–7) (4.70 ± 2.13 versus 10.87 ± 2.51, P < 0.001) compared with cerulein-treated WT mice at the same time point. Conversely, Ace2 TG mice with normal ACE expression were more resistant to SAP challenge as evidenced by a decreased inflammatory response, attenuated pathological changes and increased survival rates. These data suggest that the ACE2–ACE imbalance plays an important role in the pathogenesis of SAP and that pancreatic ACE2 is an important factor in determining the severity of SAP.
Muscles of mdx mice are known to be more susceptible to contraction-induced damage than wild-type muscle. However, it is not clear whether this is because of dystrophin deficiency or because of the abnormal branching morphology of dystrophic muscle fibres. This distinction has an important bearing on our traditional understanding of the function of dystrophin as a mechanical stabilizer of the sarcolemma. In this study, we address the question: ‘Does dystrophin-positive, regenerated muscle containing branched fibres also show an increased susceptibility to contraction-induced damage?’ We produced a model of fibre branching by injecting dystrophin-positive extensor digitorum longus muscles with notexin. The regenerated muscle was examined at 21 days postinjection. Notexin-injected muscle contained 29% branched fibres and was not more susceptible to damage from mild eccentric contractions than contralateral saline-injected control muscle. Regenerated muscles also had greater mass, greater cross-sectional area and lower specific force than control muscles. We conclude that the number of branched fibres in this regenerated muscle is below the threshold needed to increase susceptibility to damage. However, it would serve as an ideal control for muscles of young mdx mice, allowing for clearer differentiation of the effects of dystrophin deficiency from the effects of fibre regeneration and morphology.