Fastigial nucleus electrostimulation promotes axonal regeneration after experimental stroke via cAMP/PKA pathway.

Axon regeneration after cerebral ischemia in mammals is inadequate to restore function, illustrating the need to design better strategies for improving outcomes. Improvement of axon regeneration has been achieved through fastigial nucleus electrostimulation (FNS) in animal researches. However, the mechanisms underlying this neuroprotection remain poorly understood. Increasing the levels of the second messenger cyclic AMP (cAMP) enhances axon regeneration, making it an excellent candidate molecule that has therapeutic potential. In the present study, we examined the expression of cAMP signaling in ischemic brain tissues following focal cerebral ischemia. Adult rats were subjected to ischemia induced by middle cerebral artery occlusion (MCAO). A dipolar electrode was placed into the cerebellum to stimulate the cerebellar fastigial nucleus for 1 h after ischemia. Neurological deficits and the expressions of cAMP, PKA (protein kinase A) and ROCK (Rho-kinase) were determined. Axonal regeneration was measured by upregulation of growth-associated protein 43 (GAP43). The data indicated that FNS significantly enhanced axonal regeneration and motor function recovery after cerebral ischemia. FNS also significantly increased cAMP and PKA levels after ischemic brain injury. All the beneficial effects of FNS were blocked by Rp-cAMP, an antagonist of PKA. Our research suggested that the axonal regeneration conferred by FNS was likely achieved via the regulation of cAMP/PKA pathway.

Cornel Iridoid Glycoside Improves Locomotor Impairment and Decreases Spinal Cord Damage in Rats.

Purpose. This study was to investigate the effects of cornel iridoid glycoside (CIG) on spinal cord injury (SCI) in rats. Methods. The thoracic cord (at T9) of rats was injured by clip compression for 30 sec. Locomotor function was assessed using the Basso, Beattie, and Bresnahan (BBB) rating scale. Neuroanatomic stereological parameters as well as Nogo-A, p75 neurotrophin receptor (p75NTR), and ROCKII expression were measured by histological processing, immunohistochemistry, and stereological analyses. The axons passing through the lesion site were detected by BDA tracing. Results. Intragastric administration of CIG (60 and 180 mg/kg) improved the locomotor impairment at 10, 17, 24, and 31 days post-injury (dpi) compared with untreated SCI model rats. CIG treatment decreased the volume of the lesion epicenter (LEp) and increased the volume of spared tissue and the number of surviving neurons in the injured spinal cord at 31 dpi. CIG promoted the growth of BDA-positive axons and their passage through the lesion site and decreased the expression of Nogo-A, p75NTR, and ROCKII both in and around the LEp. Conclusion. CIG improved the locomotor impairment, decreased tissue damage, and downregulated the myelin-associated inhibition signaling pathway in SCI rats. The results suggest that CIG may be beneficial for SCI therapy.

Antenatal taurine improves neuronal regeneration in fetal rats with intrauterine growth restriction by inhibiting the Rho-ROCK signal pathway.

The Rho-ROCK signal pathway is an important mediator of inhibitory signals that blocks central nervous cell regeneration. Here, we investigated whether antenatal taurine improved neuronal regeneration in fetal rats with intrauterine growth restriction (IUGR) by inhibiting this pathway. Thirty pregnant rats were randomly divided into three groups: control, IUGR, and IUGR + antenatal taurine supplementation (taurine group). The mRNA levels of Ras homolog gene A (Rho A), Rho-associated coiled-coil forming protein kinase 2 (ROCK2), and proliferating cell nuclear antigen (PCNA) were detected using real-time quantitative PCR. RhoA, ROCK2 and PCNA-positive cells were counted using immunohistochemistry. Antenatal taurine supplementation decreased RhoA and Rock2 mRNA expression, increased PCNA mRNA expression, and significantly decreased RhoA, ROCK2-positive and increased PCNA-positive cell counts in IUGR fetal rat brain tissues (p < 0.05). Thus, antenatal taurine supplementation inhibited the expression of key Rho-ROCK signal molecules and improved IUGR fetal brain development.

ß-Asarone prevents autophagy and synaptic loss by reducing ROCK expression in asenescence-accelerated prone 8 mice.

ß-Asarone is an active component of the Acori graminei rhizome that is a traditional Chinese medicine clinically used in treating dementia in China. However, the cognitive effect of ß-asarone and its mechanism has remained elusive. Here, we used asenescence-accelerated prone 8 (SAMP8) mice, which mimic many of the salient features of Alzheimer׳s disease (AD), to further investigate whether modulation of the ROCK signaling pathway and/or autophagy, synaptic loss is involved in the effects of ß-asarone on learning and memory. SAMP8 mice at the age of 6 months were intragastrically administered by ß-asarone or a vehicle daily for 2 months. Senescence-accelerated-resistant (SAMR1) mice were used as the control. Our results demonstrate that autophagy and ROCK expression were increased significantly in 8 months SAMP8 mice, which were concomitant with that SAMP8 mice at the same age displayed a significant synaptic loss and cognitive deficits. The up-regulation of ROCK expression and autophage in the hippocampus of SAMP8 were significantly reduced by ß-asarone, and prevents synaptic loss and improved cognitive function of the SAMP8 mice. ß-asarone decreased neuronophagia and lipofuscin in the hippocampus of SAMP8 mice, but did not reduce Aß42 levels and malondialdehyde levels and superoxide dismutase activities. Moreover, suppression of ROCK2 by siRNA significantly reduced the effects of ß-asarone on the autophage and synaptic proteins expression in PC12 cells damage induced by Aß1-40. Taken together, ß-asarone prevents autophagy and synaptic loss by reducing ROCK expression in SAMP8 mice.

Effect of Anacyclus pyrethrum on pentylenetetrazole-induced kindling, spatial memory, oxidative stress and rho-kinase II expression in mice.

Anacyclus pyrethrum (A. pyrethrum) has been reported to exhibit anticonvulsant activity. In the present study, the effect of hydro-alcoholic extract of A. pyrethrum root (HEAP) on pentylenetetrazole (PTZ) induced kindling, spatial memory, oxidative stress and rho kinase (ROCK II) was assessed. Male albino mice (25-30 g) were used in the study. PTZ (35 mg/kg, i.p. on alternate days) was injected to induce kindling and PTZ (70 mg/kg, i.p) challenge was given 7 days post-kindling. HEAP was administered orally daily in the doses of 100, 250 and 500 mg/kg along with PTZ injections during the kindling process and continued till PTZ challenge post kindling. Spatial memory was assessed using Morris water maze test. Oxidative stress parameters [malondialdehyde (MDA) and reduced glutathione (GSH)] and ROCK II expression were estimated in whole brain at the end of the study. Pre-treatment with HEAP (250 and 500 mg/kg) showed significant increase in the myoclonic jerk latency and delay in the development of kindling. A significant decrease in mortality was observed at higher doses of HEAP (250 and 500 mg/kg). Pre-treatment with HEAP significantly increased the number of platform crossings and decreased the escape latency, as opposed to the PTZ group, thus showing protection against memory deficit. HEAP pre-treatment also attenuated the oxidative stress induced by PTZ kindling. PTZ induced kindling increased the ROCK II expression whereas, HEAP pre-treatment attenuated the increase in ROCK II expression. To conclude, HEAP pre-treatment showed antiepileptic effect and also showed protection against cognitive impairment by decreasing oxidative stress and ROCK II expression in PTZ kindled mice.

Estrogens regulate humans and rabbit epididymal contractility through the RhoA/Rho-kinase pathway.

INTRODUCTION: We have previously demonstrated that oxytocin (OT) and endothelin-1 (ET-1) peripherally regulate epididymal motility in an estrogen-dependent way. Because RhoA/Rho-kinase (ROCK) pathway is a contractile effector downstream to both OT and ET-1 receptors, we hypothesized an estrogenic modulation of OT- and ET-1-induced contraction through the up-regulation of RhoA/ROCK signaling. AIM: To evaluate the effect of changing endocrine milieu on RhoA/ROCK pathway in the epididymis. METHODS: We induced a pharmacological hypogonadotropic hypogonadism in rabbits and replaced hypogonadal animals with different sex steroids (testosterone, T, or estradiol valerate, [E(2v)]). Effects of estrogen deprivation were also evaluated in rabbits chronically treated with the P450-aromatase inhibitor letrozole. An "in vitro" model of human epididymal smooth muscle cells was established and stimulated with sex hormones (72 hours). Protein and mRNA expression and functional activity of RhoA/ROCK signaling were studied by quantitative reverse transcriptase-polymerase chain reaction, immunohistochemistry, western blot analysis, cell migration and by "in vitro" contractility studies using the ROCK inhibitor Y-27632. MAIN OUTCOME MEASURES: Effects of sex steroids on expression and functional activation of RhoA/ROCK signaling in rabbit epididymis and human epididymal smooth muscle cells. RESULTS: The relaxant effect of Y-27632 on ET-1-pre-contracted epididymal strips was significantly reduced in hypogonadal rabbits, as well as in letrozole-treated animals. T supplementation normalized T plasma levels, but not Y-27632 epididymal strip sensitivity. E(2)v not only completely restored Y-27632 responsiveness but even amplified it, indicating an estrogenic up-regulation of RhoA/ROCK pathway. Accordingly, ROCK1 protein and gene expressions were strongly induced by E(2)v but not by T. The estrogen-induced up-regulation of RhoA/ROCK signaling was confirmed in human epididymal smooth muscle cells. CONCLUSIONS: Our results suggest that estrogens regulate epididymal motility by increasing RhoA/ROCK signaling, and therefore calcium sensitivity, which tunes up responsiveness to contractile factors.

Involvement of RhoA/Rho kinase signaling in protection against monocrotaline-induced pulmonary hypertension in pneumonectomized rats by dehydroepiandrosterone.

RhoA/Rho kinase (ROCK) signaling plays a key role in the pathogenesis of experimental pulmonary hypertension (PH). Dehydroepiandrosterone (DHEA), a naturally occurring steroid hormone, effectively inhibits chronic hypoxic PH, but the responsible mechanisms are unclear. This study tested whether DHEA was also effective in treating monocrotaline (MCT)-induced PH in left pneumonectomized rats and whether inhibition of RhoA/ROCK signaling was involved in the protective effect of DHEA. Three weeks after MCT injection, pneumonectomized rats developed PH with severe vascular remodeling, including occlusive neointimal lesions in pulmonary arterioles. In lungs from these animals, we detected cleaved (constitutively active) ROCK I as well as increases in activities of RhoA and ROCK and increases in ROCK II protein expression. Chronic DHEA treatment (1%, by food for 3 wk) markedly inhibited the MCT-induced PH (mean pulmonary artery pressures after treatment with 0% and 1% DHEA were 33+/-5 and 16+/-1 mmHg, respectively) and severe pulmonary vascular remodeling in pneumonectomized rats. The MCT-induced changes in RhoA/ROCK-related protein expression were nearly normalized by DHEA. A 3-wk DHEA treatment (1%) started 3 wk after MCT injection completely inhibited the progression of PH (mean pulmonary artery pressures after treatment with 0% and 1% DHEA were 47+/-3 and 30+/-3 mmHg, respectively), and this treatment also resulted in 100% survival in contrast to 30% in DHEA-untreated rats. These results suggest that inhibition of RhoA/ROCK signaling, including the cleavage and constitutive activation of ROCK I, is an important component of the impressive protection of DHEA against MCT-induced PH in pneumonectomized rats.

Effects of San-Huang-Xie-Xin-Tang on U46619-induced increase in pulmonary arterial blood pressure.

ETHNOPHARMACOLOGICAL RELEVANCE: San-Huang-Xie-Xin-Tang (SHXT), composed of Coptidis rhizoma, Scutellariae radix and Rhei rhizoma, is traditionally used to treat hypertension. AIM OF THE STUDY: Our aim was to investigate the pharmacology effect of SHXT on a thromboxane A(2) analogue U46619-induced increase in pulmonary hypertension and protein expression in primary pulmonary smooth muscle cells (PASMCs). MATERIALS AND METHODS: Arterial blood pressure and isometric tension in the aorta and pulmonary artery of rats were measured by pressure and force transducers, respectively. Protein expressions on PASMCs were detected by Western blotting. RESULTS: SHXT significantly attenuated U46619-induced increase in arterial blood pressure. The inhibitory effect of SHXT on pulmonary arterial pressure was greater than systemic arterial pressure in U46619 treated rats. Similarly, the inhibitory effect of SHXT on U46619-induced vasoconstriction in rat pulmonary arterial rings was greater than that in aortic rings. In U46619 treated PASMCs, SHXT down-regulated expression of phosphodiesterase type 5 (PDE5), Rho-kinase (ROCK) II, cyclooxygenase-2 (COX-2) and up-regulated expression of soluble guanylyl cyclase (sGC) alpha(1) and sGCbeta(1). CONCLUSIONS: SHXT attenuated U46619-induced increase in systemic and pulmonary arterial blood pressure. Inhibition of PDE5, ROCK-II, COX-2 and stimulation of sGC may play important roles in the cardiovascular effects of SHXT.