GABA, serotonin, muscarinic receptors - their second messengers and transcription factors functional regulation in the brain regions of hypoxic neonatal rats: Resuscitation with glucose, oxygen and epinephrine

The present study was designed to investigate the protective effect of glucose, oxygen and epinephrine resuscitation on impairment in the functional role of GABAergic, serotonergic, muscarinic receptors, PLC, BAX, SOD, CAT and GPx expression in the brain regions of hypoxia induced neonatal rats. Also, the role of hormones - Triiodothyronine (T3) and insulin, second messengers – cAMP, cGMP and IP3 and transcription factors – HIF and CREB in the regulation of neonatal hypoxia and its resuscitation methods were studied. Behavioural studies were conducted to evaluate the motor function and cognitive deficit in one month old control and experimental rats. The efficient and timely supplementation of glucose plays a crucial role in correcting the molecular changes due to hypoxia, oxygen and epinephrine. The sequence of glucose, epinephrine and oxygen administration at the molecular level is an important aspect of the study. The additive neuronal damage effect due to oxygen and epinephrine treatment is another important observation. The corrective measures by initial supply of glucose to hypoxic neonatal rats showed from the molecular study when brought to practice will lead to healthy intellectual capacity during the later developmental stages, which has immense clinical significance in neonatal care.

Potential Role of Growth Hormone in Impairment of Insulin Signaling in Skeletal Muscle, Adipose Tissue, and Liver of Rats Chronically Treated with Arginine

We have shown that rats chronically treated with Arginine (Arg), although normoglycemic, exhibit hyperinsulinemia and decreased blood glucose disappearance rate after an insulin challenge. Attempting to investigate the processes underlying these alterations, male Wistar rats were treated with Arg (35 mg/d), in drinking water, for 4 wk. Rats were then acutely stimulated with insulin, and the soleus and extensorum digitalis longus muscles, white adipose tissue (WAT), and liver were excised for total and/or phosphorylated insulin receptor (IR), IR substrate 1/2, Akt, Janus kinase 2, signal transducer and activator of transcription (STAT) 1/3/5, and p85 alpha/55 alpha determination. Muscles and WAT were also used for plasma membrane (PM) and microsome evaluation of glucose transporter (GLUT) 4 content. Pituitary GH mRNA, GH, and liver IGF-I mRNA expression were estimated. It was shown that Arg treatment: 1) did not affect phosphotyrosine-IR, whereas it decreased phosphotyrosine-IR substrate 1/2 and phosphoserine-Akt content in all tissues studied, indicating that insulin signaling is impaired at post-receptor level; 2) decreased PM GLUT4 content in both muscles and WAT; 3) increased the pituitary GH mRNA, GH, and liver IGF-I mRNA expression, the levels of phosphotyrosine-STAT5 in both muscles, phosphotyrosine-Janus kinase 2 in extensorum digitalis longus, phosphotyrosine-STAT3 in liver, and WAT as well as total p85 alpha in soleus, indicating that GH signaling is enhanced in these tissues; and 4) increased p55 alpha total content in muscles, WAT, and liver. The present findings provide the molecular mechanisms by which insulin resistance and, by extension, reduced GLUT4 content in PM of muscles and WAT take place after chronic administration of Arg, and further suggest a putative role for GH in its genesis, considering its diabetogenic effect. (Endocrinology 150: 2080-2086, 2009)

Proteomic Analysis of Urine in Rats Chronically Exposed to Fluoride

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Proteomic analysis of kidney in rats chronically exposed to fluoride

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Participation of the dorsal periaqueductal grey matter in the hypoxic ventilatory response in unanaesthetized rats

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Proteomic Analysis of Gastrocnemius Muscle in Rats with Streptozotocin-Induced Diabetes and Chronically Exposed to Fluoride

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Catecholaminergic (CA) neurons modulate hypoxic ventilatory response in newborn rats (P7-8)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Hypoxic and hypercapnic ventilatory responses in rats with polycystic ovaries

In female rats, a single injection of estradiol valerate (EV) results in effects that are similar to those observed in women with polycystic ovary syndrome (PCOS). We hypothesized that EV-induced PCOS affects breathing control based on evidence showing an influence of sex hormones on ventilation. To test this hypothesis, we studied the effects of EV treatment on the ventilation of female rats in air, in 7% CO2 and in 7% O2, at 30, 45 and 60 days after EV injection. The group examined 30 days after EV treatment showed a 61% reduction in the hypercapnic ventilatory response compared to the control group. Basal ventilation, hypoxic ventilatory response, and body temperature were not affected. These results, suggest that the hormonal changes observed in PCOS may result in a temporary inhibition of the central chemoreflex but do not influence basal ventilation or the hypoxic peripheral chemoreflex.

Intracisternal application of endotoxin enhances the susceptibility to subsequent hypoxic-ischemic brain damage in neonatal rats.

Perinatal brain damage is associated not only with hypoxic-ischemic insults but also with intrauterine inflammation. A combination of antenatal inflammation and asphyxia increases the risk of cerebral palsy >70 times. The aim of the present study was to determine the effect of intracisternal (i.c.) administration of endotoxin [lipopolysaccharides (LPS)] on subsequent hypoxic-ischemic brain damage in neonatal rats. Seven-day-old Wistar rats were subjected to i.c. application of NaCl or LPS (5 microg/pup). One hour later, the left common carotid artery was exposed through a midline neck incision and ligated with 6-0 surgical silk. After another hour of recovery, the pups were subjected to a hypoxic gas mixture (8% oxygen/92% nitrogen) for 60 min. The animals were randomized to four experimental groups: 1) sham control group, left common carotid artery exposed but not ligated (n = 5); 2) LPS group, subjected to i.c. application of LPS (n = 7); 3) hypoxic-ischemic study group, i.c. injection of NaCl and exposure to hypoxia after ligation of the left carotid artery (n = 17); or 4) hypoxic-ischemic/LPS study group, i.c. injection of LPS and exposure to hypoxia after ligation of the left carotid artery (n = 19). Seven days later, neonatal brains were assessed for neuronal cell damage. In a second set of experiments, rat pups received an i.c. injection of LPS (5 microg/pup) and were evaluated for tumor necrosis factor-alpha expression by immunohistochemistry. Neuronal cell damage could not be observed in the sham control or in the LPS group. In the hypoxic-ischemic/LPS group, neuronal injury in the cerebral cortex was significantly higher than in animals that were subjected to hypoxia/ischemia after i.c. application of NaCl. Injecting LPS intracisternally caused a marked expression of tumor necrosis factor-alpha in the leptomeninges. Applying LPS intracisternally sensitizes the immature rat brain to a subsequent hypoxic-ischemic insult.

Microelectrode Implants for Spinal Cord Stimulation in Rats

Paralysis is a debilitating condition afflicting millions of people across the globe, and is particularly deleterious to quality of life when motor function of the legs is severely impaired or completely absent. Fortunately, spinal cord stimulation has shown great potential for improving motor function after spinal cord injury and other pathological conditions. Many animal studies have shown stimulation of the neural networks in the spinal cord can improve motor ability so dramatically that the animals can even stand and step after a complete spinal cord transaction.

This thesis presents work to successfully provide a chronically implantable device for rats that greatly enhances the ability to control the site of spinal cord stimulation. This is achieved through the use of a parylene-C based microelectrode array, which enables a density of stimulation sites unattainable with conventional wire electrodes. While many microelectrode devices have been proposed in the past, the spinal cord is a particularly challenging environment due to the bending and movement it undergoes in a live animal. The developed microelectrode array is the first to have been implanted in vivo while retaining functionality for over a month. In doing so, different neural pathways can be selectively activated to facilitate standing and stepping in spinalized rats using various electrode combinations, and important differences in responses are observed.

An engineering challenge for the usability of any high density electrode array is connecting the numerous electrodes to a stimulation source. This thesis develops several technologies to address this challenge, beginning with a fully passive implant that uses one wire per electrode to connect to an external stimulation source. The number of wires passing through the body and the skin proved to be a hazard for the health of the animal, so a multiplexed implant was devised in which active electronics reduce the number of wires. Finally, a fully wireless implant was developed. As these implants are tested in vivo, encapsulation is of critical importance to retain functionality in a chronic experiment, especially for the active implants, and it was achieved without the use of costly ceramic or metallic hermetic packaging. Active implants were built that retained functionality 8 weeks after implantation, and achieved stepping in spinalized rats after just 8-10 days, which is far sooner than wire-based electrical stimulation has achieved in prior work.

Pretreatment with Resveratrol Prevents Neuronal Injury and Cognitive Deficits Induced by Perinatal Hypoxia-Ischemia in Rats

Despite advances in neonatal care, hypoxic-ischemic brain injury is still a serious clinical problem, which is responsible for many cases of perinatal mortality, cerebral palsy, motor impairment and cognitive deficits. Resveratrol, a natural polyphenol with important anti-oxidant and anti-inflammatory properties, is present in grapevines, peanuts and pomegranates. The aim of the present work was to evaluate the possible neuroprotective effect of resveratrol when administered before or immediately after a hypoxic-ischemic brain event in neonatal rats by analyzing brain damage, the mitochondrial status and long-term cognitive impairment. Our results indicate that pretreatment with resveratrol protects against brain damage, reducing infarct volume, preserving myelination and minimizing the astroglial reactive response. Moreover its neuroprotective effect was found to be long lasting, as behavioral outcomes were significantly improved at adulthood. We speculate that one of the mechanisms for this neuroprotection may be related to the maintenance of the mitochondrial inner membrane integrity and potential, and to the reduction of reactive oxygen species. Curiously, none of these protective features was observed when resveratrol was administered immediately after hypoxia-ischemia.

Effects of Naotan Pill on repair of neural cells and cognitive disorders in juvenile rats following hypoxia and ischemia

BACKGROUND: Hypoxia and ischemia induce neuronal damage, decreased neuronal numbers and synaptophysin levels, and deficits in learning and memory functions. Previous studies have shown that lycium barbarum polysaccharide, the most effective component of barbary wolfberry fruit, has protective effects on neural cells in hypoxia-ischemia. OBJECTIVE: To investigate the effects of Naotan Pill on glutamate-treated neural cells and on cognitive function in juvenile rats following hypoxia-ischemia. DESIGN, TIME AND SETTING: The randomized, controlled, in vivo study was performed at the Cell Laboratory of Lanzhou University, Lanzhou Institute of Modern Physics of Chinese Academy of Sciences, and Department of Traditional Chinese Medicine of Gansu Provincial Rehabilitation Center Hospital, China from December 2005 to August 2006. The cellular neurobiology, in vitro experiment was conducted at the Institute of Human Anatomy, Histology, Embryology and Neuroscience, School of Basic Medical Sciences, Lanzhou University, and Department of Traditional Chinese Medicine of Gansu Provincial Rehabilitation Center Hospital, China from March 2007 to January 2008. MATERIALS: Naotan Pill, composed of barbary wolfberry fruit, danshen root, grassleaf sweetflag rhizome, and glossy privet fruit, was prepared by Gansu Provincial Rehabilitation Center, China. Rabbit anti-synaptophysin, choline acetyl transferase polyclonal antibody, streptavidin-biotin complex kit and diaminobenzidine kit (Boster, Wuhan, China), as well as glutamate (Hualian, Shanghai, China) were used in this study. METHODS: Cortical neural cells were isolated from neonatal Wistar rats. Neural cell damage models were induced using glutamate, and administered Naotan Pill prior to and following damage. A total of 54 juvenile Wistar rats were equally and randomly assigned into model, Naotan Pill, and sham operation groups. The left common carotid artery was ligated, and then rat models of hypoxic-ischemic injury were assigned to the model and Naotan Pill groups. At 2 days following model induction, rats in the Naotan Pill group were administered Naotan Pill suspension for 21 days. In the model and sham operation groups, rats received an equal volume of saline. MAIN OUTCOME MEASURES: Neural cell morphology was observed using an inverted phase contrast microscope. Survival rate of neural cells was measured by MTT assay. Synaptophysin and choline acetyl transferase expression was observed in the hippocampal CA1 region of juvenile rats using immunohistochemistry. Cognitive function was tested by the Morris water maze. RESULTS: Pathological changes were detected in glutamate-treated neural cells. Neural cell morphology remained normal after Naotan Pill intervention. Absorbance and survival rate of neural cells were significantly greater following Naotan Pill intervention, compared to glutamate-treated neural cells (P < 0.05). Synaptophysin and choline acetyl transferase expression was lowest in the hippocampal CA1 region in the model group and highest in the sham operation group. Significant differences among groups were observed (P < 0.05). Escape latency and swimming distance were significantly longer in the model group compared to the Naotan Pill group (P < 0.05). CONCLUSION: Naotan Pill exhibited protective and repair effects on glutamate-treated neural cells. Naotan Pill upregulated synaptophysin and choline acetyl transferase expression in the hippocampus and improved cognitive function in rats following hypoxia-ischemia.

Anti-hypotensive treatment and endothelin blockade synergistically antagonize exercise fatigue in rats under simulated high altitude.

Rapid ascent to high altitude causes illness and fatigue, and there is a demand for effective acute treatments to alleviate such effects. We hypothesized that increased oxygen delivery to the tissue using a combination of a hypertensive agent and an endothelin receptor A antagonist drugs would limit exercise-induced fatigue at simulated high altitude. Our data showed that the combination of 0.1 mg/kg ambrisentan with either 20 mg/kg ephedrine or 10 mg/kg methylphenidate significantly improved exercise duration in rats at simulated altitude of 4,267 m, whereas the individual compounds did not. In normoxic, anesthetized rats, ephedrine alone and in combination with ambrisentan increased heart rate, peripheral blood flow, carotid and pulmonary arterial pressures, breathing rate, and vastus lateralis muscle oxygenation, but under inspired hypoxia, only the combination treatment significantly enhanced muscle oxygenation. Our results suggest that sympathomimetic agents combined with endothelin-A receptor blockers offset altitude-induced fatigue in rats by synergistically increasing the delivery rate of oxygen to hypoxic muscle by concomitantly augmenting perfusion pressure and improving capillary conductance in the skeletal muscle. Our findings might therefore serve as a basis to develop an effective treatment to prevent high-altitude illness and fatigue in humans.