Evaluation of the Cardiometabolic Disorders after Spinal Cord Injury in Mice.

Changes in cardiometabolic functions contribute to increased morbidity and mortality after chronic spinal cord injury. Despite many advancements in discovering SCI-induced pathologies, the cardiometabolic risks and divergences in severity-related responses have yet to be elucidated. Here, we examined the effects of SCI severity on functional recovery and cardiometabolic functions following moderate (50 kdyn) and severe (75 kdyn) contusions in the thoracic-8 (T8) vertebrae in mice using imaging, morphometric, and molecular analyses. Both severities reduced hindlimbs motor functions, body weight (g), and total body fat (%) at all-time points up to 20 weeks post-injury (PI), while only severe SCI reduced the total body lean (%). Severe SCI increased liver echogenicity starting from 12 weeks PI, with an increase in liver fibrosis in both moderate and severe SCI. Severe SCI mice showed a significant reduction in left ventricular internal diameters and LV volume at 20 weeks PI, associated with increased LV ejection fraction as well as cardiac fibrosis. These cardiometabolic dysfunctions were accompanied by changes in the inflammation profile, varying with the severity of the injury, but not in the lipid profile nor cardiac or hepatic tyrosine hydroxylase innervation changes, suggesting that systemic inflammation may be involved in these SCI-induced health complications.

Maternal obesity in sheep impairs foetal hepatic mitochondrial respiratory chain capacity.

BACKGROUND: Changes in the nutritional environment in utero induced by maternal obesity (MO) lead to foetal metabolic dysfunction predisposing offspring to later-life metabolic diseases. Since mitochondria play a crucial role in hepatic metabolism and function, we hypothesized that MO prior to conception and throughout pregnancy programmes foetal sheep liver mitochondrial phenotype. MATERIAL AND METHODS: Ewes ate an obesogenic diet (150% requirements; MO), or 100% requirements (CTR), from 60 days prior to conception. Foetal livers were removed at 0.9 gestation. We measured foetal liver mitochondrial DNA copy number, activity of superoxide dismutase, cathepsins B and D and selected protein content, total phospholipids and cardiolipin and activity of mitochondrial respiratory chain complexes. RESULTS: A significant decrease in activities of mitochondrial complexes I, II-III and IV, but not aconitase, was observed in MO. In the antioxidant machinery, there was a significant increase in activity of total superoxide dismutase (SOD) and SOD2 in MO. However, no differences were found regarding autophagy-related protein content (p62, beclin-I, LC3-I, LC3-II and Lamp2A) and cathepsin B and D activities. A 21.5% decrease in total mitochondrial phospholipid was observed in MO. CONCLUSIONS: The data indicate that MO impairs foetal hepatic mitochondrial oxidative capacity and affects total mitochondrial phospholipid content. In addition, MO affects the regulation of foetal liver redox pathways, indicating metabolic adaptations to the higher foetal lipid environment. Consequences of in utero programming of foetal hepatic metabolism may persist and compromise mitochondrial bioenergetics in later life, and increase susceptibility to metabolic diseases.

A heretical view: rather than a solely placental protective function, placental 11ß hydroxysteroid dehydrogenase 2 also provides substrate for fetal peripheral cortisol synthesis in obese pregnant ewes.

Exposure to glucocorticoid levels higher than appropriate for current developmental stages induces offspring metabolic dysfunction. Overfed/obese (OB) ewes and their fetuses display elevated blood cortisol, while fetal Adrenocorticotropic hormone (ACTH) remains unchanged. We hypothesized that OB pregnancies would show increased placental 11ß hydroxysteroid dehydrogenase 2 (11ß-HSD2) that converts maternal cortisol to fetal cortisone as it crosses the placenta and increased 11ß-HSD system components responsible for peripheral tissue cortisol production, providing a mechanism for ACTH-independent increase in circulating fetal cortisol. Control ewes ate 100% National Research Council recommendations (CON) and OB ewes ate 150% CON diet from 60 days before conception until necropsy at day 135 gestation. At necropsy, maternal jugular and umbilical venous blood, fetal liver, perirenal fat, and cotyledonary tissues were harvested. Maternal plasma cortisol and fetal cortisol and cortisone were measured. Fetal liver, perirenal fat, cotyledonary 11ß-HSD1, hexose-6-phosphate dehydrogenase (H6PD), and 11ß-HSD2 protein abundance were determined by Western blot. Maternal plasma cortisol, fetal plasma cortisol, and cortisone were higher in OB vs. CON (p < 0.01). 11ß-HSD2 protein was greater (p < 0.05) in OB cotyledonary tissue than CON. 11ß-HSD1 abundance increased (p < 0.05) in OB vs. CON fetal liver and perirenal fat. Fetal H6PD, an 11ß-HSD1 cofactor, also increased (p < 0.05) in OB vs. CON perirenal fat and tended to be elevated in OB liver (p < 0.10). Our data provide evidence for increased 11ß-HSD system components responsible for peripheral tissue cortisol production in fetal liver and adipose tissue, thereby providing a mechanism for an ACTH-independent increase in circulating fetal cortisol in OB fetuses.

A Review of Maternal Nutrition during Pregnancy and Impact on the Offspring through Development: Evidence from Animal Models of Over- and Undernutrition.

Similarities in offspring phenotype due to maternal under- or over-nutrition during gestation have been observed in studies conducted at University of Wyoming. In these studies, ewes were either nutrient-restricted (NR) from early to mid-gestation, or fed an obesogenic diet (MO) from preconception through term. Offspring necropsies occurred at mid-gestation, late-gestation, and after parturition. At mid gestation, body weights of NR fetuses were ~30% lighter than controls, whereas MO fetuses were ~30% heavier than those of controls. At birth, lambs born to NR, MO, and control ewes exhibited similar weights. This was a consequence of accelerated fetal growth rates in NR ewes, and reduced fetal growth rates in MO ewes in late gestation, when compared to their respective controls. These fetal growth patterns resulted in remarkably similar effects of increased susceptibility to obesity, cardiovascular disease, and glucose intolerance in offspring programmed mostly during fetal stages of development. These data provide evidence that maternal under- and over-nutrition similarly induce the development of the same cadre of physical and metabolic problems in postnatal life.

Long-term neural regeneration following injury to the peroneal branch of the sciatic nerve in sheep.

Peripheral nerves (PNs) are frequently injured as a result of trauma or disease. Development of therapies to regenerate PNs requires the use of animal models, typically beginning in rodents and progressing to larger species. There are several large animal models of PN regeneration that each has their benefits and drawbacks. Sheep have been used in PN studies due to their similarities in body weight to humans and the ease and lesser expense in their care and housing relative to other species. We have investigated the use of sheep for studies of PN regeneration and have developed and tested an injury model in the peroneal branch of the sciatic nerve. Three experimental groups were tested on mature sheep: a bisection; a 5-cm reverse autograft; and sham surgery. Protocols were developed for the post-operative care for animals with this injury, and regeneration was tracked for extended time points via compound muscle action potentials (CMAPs) and endpoint assessments of nerve morphometry, muscle mass and muscle fibrosis. Results indicate the practical viability of this PN injury model and show distinctions in the degree and rate of regeneration between bisection and reverse autograft that persisted 14 months. This long-term study shows bisections lead to significantly improved CMAPS and muscle mass and lesser muscle fibrosis as compared to reverse autograft. The persistence of these discernable changes between two relatively similar experimental groups out to extended time points is an indication of the sensitivity of this nerve section and its potential applicability for comparative studies.

A Long-Term Pilot Study on Sex and Spinal Cord Injury Shows Sexual Dimorphism in Functional Recovery and Cardio-Metabolic Responses.

More than a quarter of a million individuals in the US live with spinal cord injury (SCI). SCI disrupts neural circuitry to vital organs in the body. Despite severe incidences of long-term peripheral complications from SCI, the cardio-metabolic consequences and divergences in sex-related responses are not well described. We examined the effects of SCI on functional recovery, cardiac structure and function, body composition, and glucose metabolism on adult female and male Sprague Dawley (SD) rats. SCI was induced at T10 via contusion. Measured outcomes include behavioral assessment, body weight, dual-energy X-ray absorptiometry (DEXA) for body composition, echocardiography for cardiac structure and function, intraperitoneal glucose tolerance test (IPGTT) for glucose metabolism, insulin tolerance test (ITT), and histology of cardiac structure at the endpoint. There was a decrease in body fat percentage in both sexes, with SCI females disproportionately affected in percent body fat change. Left ventricular internal diameter during systole (LVIDs) was decreased in SCI females more than in SCI males. No significant differences in glucose metabolism were observed up to 20 weeks post-injury (PI). These data show significant cardio-metabolic differences as a consequence of SCI and, furthermore, that sex is an underlying factor in these differences.

Rapid Communication: Reduced maternal nutrition during early- to mid-gestation elevates newborn lamb plasma cortisol concentrations and eliminates the neonatal leptin surge.

Human epidemiological and animal studies show that maternal nutrient reduction (MNR) and maternal overnutrition/obesity (MO) alter fetal growth and development, predisposing offspring (F1) to endocrine and appetite dysregulation. Compared to F1 of control-fed ewes, F1 of MO ewes display hypercortisolemia at birth and fail to exhibit the neonatal leptin surge implicated in lifelong appetite regulation. Here, we determined if MNR also elevates newborn lamb plasma cortisol and eliminates the neonatal leptin surge. Starting 30 d prior to conception, nulliparous control (CON, n = 6) ewes ate 100% NRC recommendations through parturition. Nutrient-reduced (NR, n = 6) ewes ate a CON diet through day 27 of gestation. From gestational days 28 to 78, NR ewes ate 50% of the CON diet before realimentation to 100% NRC recommendations. Jugular blood was collected daily from lambs from birth (day 0) through postnatal day 10, to determine plasma cortisol and leptin. Newborn NR plasma cortisol concentrations were increased (P < 0.0001) vs. CON and were similar to concentrations in MO lambs. Plasma leptin concentrations were similar between groups through postnatal day 7. The leptin surge, seen in CON lambs on postnatal days 8 to 10 was not present in NR lambs. These data show that, similar to MO lambs, early pregnancy MNR elevates newborn lamb plasma cortisol and eliminates the neonatal leptin surge. In the light of the similar elevation of neonatal cortisol in MNR and MO lambs, we conclude that cortisol plays a central role in regulating the neonatal lamb leptin surge.

Toluidine Blue Staining of Resin-Embedded Sections for Evaluation of Peripheral Nerve Morphology.

Peripheral nerves extend throughout the body, innervating target tissues with motor or sensory axons. Due to widespread distribution, peripheral nerves are frequently damaged because of trauma or disease. As methods and strategies have been developed to assess peripheral nerve injury in animal models, function and regeneration, analyzing the morphometry of the peripheral nerve has become an essential terminal outcome measurement. Toluidine blue staining of nerve cross sections obtained from resin embedded nerve sections is a reproducible method for qualitative and quantitative assessments of peripheral nerves, enabling visualization of morphology number of axons and degree of myelination. This technique, as with many other histological methods, can be difficult to learn and master using standard written protocols. The intent of this publication is therefore to accentuate written protocols for toluidine blue staining of peripheral nerves with videography of the method, using sciatic nerves harvested from rats. In this protocol, we describe in vivo peripheral nerve fixation and collection of the tissue, and post-fixation with 2% osmium tetroxide, embedding of nerves in epoxy resin, and ultramicrotome sectioning of nerves to 1-2µm thickness. Nerve sections then transferred to a glass slide and stained with toluidine blue, after which they are quantitatively and qualitatively assessed. Examples of the most common problems are shown, as well as steps for mitigating these issues.

Maternal obesity in the ewe increases cardiac ventricular expression of glucocorticoid receptors, proinflammatory cytokines and fibrosis in adult male offspring.

Obesity during human pregnancy predisposes offspring to obesity and cardiovascular disease in postnatal life. In a sheep model of maternal overnutrition/obesity we have previously reported myocardial inflammation and fibrosis, as well as cardiac dysfunction in late term fetuses, in association with chronically elevated blood cortisol. Significant research has suggested a link between elevated glucocorticoid exposure in utero and hypertension and cardiovascular disease postnatally. Here we examined the effects of maternal obesity on myocardial inflammation and fibrosis of their adult offspring. Adult male offspring from control (CON) mothers fed 100% of National Research Council (NRC) recommendations (n = 6) and male offspring from obese mothers (MO) fed 150% NRC (n = 6), were put on a 12-week ad libitum feeding challenge then necropsied. At necropsy, plasma cortisol and left and right ventricular thickness were markedly increased (P<0.05) in adult male MO offspring. Myocardial collagen content and collagen-crosslinking were greater (P<0.05) in MO offspring compared to CON offspring in association with increased mRNA and protein expression of glucocorticoid receptors (GR). No group difference was found in myocardial mineralocorticoids receptor (MR) protein expression. Further, mRNA expression for the proinflammatory cytokines: cluster of differentiation (CD)-68, transforming growth factor (TGF)-ß1, and tumor necrosis factor (TNF)-α were increased (P < 0.05), and protein expression of CD-68, TGF-ß1, and TNF-α tended to increase (P<0.10) in MO vs. CON offspring. These data provide evidence for MO-induced programming of elevated plasma cortisol and myocardial inflammation and fibrosis in adult offspring potentially through increased GR.