Ian Appleton

Neuroprotective effects of (–)-epigallocatechin gallate following hypoxia-ischemia-induced brain damage: novel mechanisms of action

(−)‐Epigallocatechin gallate (EGCG) is a potent antioxidant that is neuroprotective against ischemia‐induced brain damage. However, the neuroprotective effects and possible mechanisms of action of EGCG after hypoxia‐ischemia (HI) have not been investigated. Therefore, we used a modified “Levine” model of HI to determine the effects of EGCG. Wistar rats were treated with either 0.9% saline or 50 mg/kg EGCG daily for 1 day and 1 h before HI induction and for a further 2 days post‐HI. At 26‐days‐old, both groups underwent permanent left common carotid artery occlusion and exposure to 8% oxygen/92% nitrogen atmosphere for 1 h. Histological assessment showed that EGCG significantly reduced infarct volume (38.0±16.4 mm3) in comparison to HI + saline (99.6±15.6 mm3). In addition, EGCG significantly reduced total (622.6±85.8 pmol l‐[3H]citrulline/30 min/mg protein) and inducible nitric oxide synthase (iNOS) activity (143.2±77.3 pmol l‐[3H]citrulline/30 min/mg protein) in comparison to HI+saline controls (996.6±113.6 and 329.7±59.6 pmol l‐[3H]citrulline/30 min/mg protein for total NOS and iNOS activity, respectively). Western blot analysis demonstrated that iNOS protein expression was also reduced. In contrast, EGCG significantly increased endothelial and neuronal NOS protein expression compared with HI controls. EGCG also significantly preserved mitochondrial energetics (complex I‐V) and citrate synthase activity. This study demonstrates that the neuroprotective effects of EGCG are, in part, due to modulation of NOS isoforms and preservation of mitochondrial complex activity and integrity. We therefore conclude that the in vivo neuroprotective effects of EGCG are not exclusively due to its antioxidant effects but involve more complex signal transduction mechanisms.

Cerebral hypoxia-ischemia and middle cerebral artery occlusion induce expression of the cannabinoid CB2 receptor in the brain

Until recently the cannabinoid CB2 receptor was believed to be absent from the central nervous system. In this study we have identified CB2 expressing cells that appear in the rat brain following stroke and hypoxic-ischemia. At 3 days following surgery CB2-positive macrophages, deriving from resident microglia and/or invading monocytes appear on the lesioned side of the brain. By day 7, a mixed population of CB2-positive cells is present. Microglia-derived macrophages are the key cells in the first stages of brain inflammation, and a pivotal step in the neurodegeneration that follows the acute stage of injury. Thus, CB2 may be important in the brain during injury, and in inflammatory neurodegenerative disorders. The presence of CB2-positive cells in the brain following stroke may provide a novel strategy for cannabinoid-mediated intervention into stroke induced neurodegeneration without the psychoactive effects of CB1 receptor stimulation.

Effects of Epicatechin Gallate on Wound Healing and Scar Formation in a Full Thickness Incisional Wound Healing Model in Rats

Catechins are naturally occurring polyphenolic compounds with putative anti-inflammatory, antioxidant and free radical scavenging effects in vitro. However, their potential effects in vivo have not been established. Therefore we have investigated the effects of the catechin epicatechin gallate (ECG), on scar formation in a full thickness incisional model of wound healing in rats. ECG showed a significant improvement in the quality of scar formation both in terms of maturity and orientation of the collagen fibers. An increase in inducible nitric oxide synthase and cyclooxygenase-2 and a decrease in arginase-I activity and protein levels were observed at earlier time points. In addition, an increase in the number of new blood vessels was observed in the ECG-treated group. This correlated with the protein levels of vascular endothelial growth factor, the most potent angiogenic protein known. This study has therefore demonstrated, for the first time, that catechins, namely ECG, can significantly improve the quality of wound healing and scar formation. These effects may in part be due to an acceleration of the angiogenic response and an up-regulation of the enzymes nitric oxide synthase and cyclooxygenase.

Regional variations and age-related changes in nitric oxide synthase and arginase in the sub-regions of the hippocampus

L-arginine can be metabolised by nitric oxide synthase (NOS) with the formation of L-citrulline and nitric oxide (NO), or arginase with the production of L-ornithine and urea. In contrast to studies showing a potential involvement of NOS/NO in the aging process, the role of arginase has not been well documented. The present study investigates for the first time the regional variations and age-related changes in both NOS and arginase in sub-regions of the hippocampus. In young adult rats, although the total NOS activity was not significantly different across the hippocampal CA1, CA2/3 and the dentate gyrus (DG) sub-regions, the total arginase activity showed a clear regional variation with the highest level in DG. Western blotting revealed that the highest levels of neuronal NOS (nNOS) and endothelial NOS (eNOS) proteins were located in CA1. Arginase I is expressed at a very low level in the brain (the whole hippocampus) as compared with the liver. By contrast, arginase II protein shows an extremely high expression in the brain with little or no expression in the liver. There was no regional variation in arginase I or arginase II protein expression across the sub-regions of the hippocampus. When a comparison was made between young (4-month-old) and aged (24-month-old) rats, a significant increase in total NOS activity was found in DG and significant decreases in arginase activity were observed in the CA1 and CA2/3 regions in the aged animals. Western blotting further revealed a dramatic decrease in eNOS protein expression in aged CA2/3 with no age-associated changes in nNOS, arginase I and II protein expression in any region examined. Interestingly, evidence of activity or protein expression of the inducible isoform of NOS (iNOS) was not detected in any tissue from either group. The present results, in conjunction with previous findings, support the contribution of NOS/NO to aging but question the involvement of iNOS in the normal aging process. Region-specific changes in arginase suggest that this enzyme may also contribute to aging.

Neuroprotective effects of spermine following hypoxic-ischemic-induced brain damage: A mechanistic study

The polyamines (spermine, putrescine, and spermidine) can have neurotoxic or neuroprotective properties in models of neurodegeneration. However, assessment in a model of hypoxia– ischemia (HI) has not been defined. Furthermore, the putative mechanisms of neuroprotection have not been elucidated. Therefore, the present study examined the effects of the polyamines in a rat pup model of HI and determined effects on key enzymes involved in inflammation, namely, nitric oxide synthase (NOS) and arginase. In addition, effects on mitochondrial function were investigated. The polyamines or saline were administered i.p. at 10mg/kg/day for 6 days post‐HI. Histological assessment 7 days post‐HI revealed that only spermine significantly (P<0.01) reduced infarct size from 46.14 ± 10.4mm3 (HI + saline) to 4.9 ± 2.7 mm3. NOS activity was significantly increased following spermine treatment in the left (ligated) hemisphere compared with nonintervention controls (P<0.01) and HI + saline (P<0.05). In contrast, spermine decreased arginase activity compared with HI + saline but was still significantly elevated in comparison to nonintervention controls (P<0.01). Assessment of mitochondrial function in the HI + saline group, revealed significant and extensive damage to complex‐I (P<0.01) and IV (P<0.001) and loss of citrate synthase activity (P<0.05). No effect on complex II‐III was observed. Spermine treatment significantly prevented all these effects. This study has therefore confirmed the neuroprotective effects of spermine in vivo. However, for the first time, we have shown that this effect may, in part, be due to increased NOS activity and preservation of mitochondrial function.

Potential involvement of NOS and arginase in age-related behavioural impairments.

The present study investigated age-related changes in nitric oxide synthase (NOS) and arginase, which shares a substrate with NOS, in the hippocampus and parahippocampal region and the relationship between NOS/arginase and age-associated behavioural impairments. Aged rats (24 months old) displayed reduced exploratory activity, enhanced anxiety, poorer spatial learning and memory, and impaired object recognition memory relative to the young adults (4 months old). There were significant increases in total NOS activity in the aged hippocampus and perirhinal, postrhinal and temporal cortices and a dramatic decrease in endothelial NOS expression in the aged postrhinal cortex. Activity and protein expression of inducible NOS were not detected in any region from either group and a significant increase in total arginase activity was found in the aged perirhinal cortex. Multiple regression analysis revealed significant correlations between NOS/arginase and behavioural measures in both groups. The present findings provide further support for a contribution of nitric oxide to the normal aging process and suggest a potential involvement of arginase in aging and learning and memory.

Expression of nitric oxide synthase isoforms in human liver cirrhosis.

Several mediators of systemic vasodilatation in liver cirrhosis have been reported. Among these is nitric oxide (NO), which has been proposed as one of the main mediators. In this study, sera and liver biopsies were analysed from 15 patients with clinically and pathologically diagnosed liver cirrhosis. In addition, sera from seven and liver biopsies from three healthy controls were used. Serum levels of nitrite (the end product of NO) were measured using the Griess reaction and the expression of the inducible nitric oxide synthase (iNOS) and constitutive nitric oxide synthase (ecNOS) proteins was investigated using immunohistochemistry. This study shows that serum nitrite levels (94 ± 9.8 µmol/l) in cirrhotic patients were significantly (p < 0.05) increased in comparison with the controls (36.6 ± 11.03 µmol/l). iNOS was completely absent from the control group but was highly expressed in the livers from the cirrhotic group. iNOS was seen mainly in the inflammatory cells infiltrating the portal tracts, blood monocyte‐like cells, hepatocytes, sinusoidal cells, and endothelial cells. However, expression of ecNOS was only seen in the vascular endothelial cells of both the control and the cirrhotic groups, but was much higher in the latter. It is therefore clear that NO is augmented in cirrhotic patients and it is mainly produced by induction of iNOS. Moreover, NO up‐regulation is dependent on the inflammatory stage of liver cirrhosis. ecNOS production could be a normal chronic adaptation mechanism of the endothelium to the chronically increased splanchnic blood flow secondary to portal hypertension. In the near future, the appropriate inhibition of NO activity by using NOS‐active agents may provide a novel strategy for the treatment of patients with liver cirrhosis. Copyright

Age-related changes in nitric oxide synthase and arginase in the rat prefrontal cortex

Increasing evidence suggests that nitric oxide (NO), generated by nitric oxide synthase (NOS) from l-arginine, plays an important role in the ageing process. The present study, for the first time, investigates age-related changes in NOS and arginase, an enzyme that shares a common substrate with NOS, in the prefrontal cortex of rats assessed with and without prior behavioural testing. A significant increase in total NOS activity was found in the prefrontal cortex in aged (24-month-old) as compared with young (4-month-old) rats. Western blotting revealed that there were no significant differences between young and aged rats in neuronal NOS (nNOS) and endothelial NOS (eNOS) protein expression. Inducible isoform of NOS (iNOS), in terms of activity and protein expression, was not detected in either group. Total arginase activity and arginase I and II protein expression did not differ between the young and aged groups. The present findings support the contribution of NOS/NO to ageing but question the importance of iNOS in the normal ageing process.

Clomethiazole: mechanisms underlying lasting neuroprotection following hypoxia-ischemia

Damage after hypoxia‐ischemia (HI) is observed in both cortical and subcortical regions. In this study, we employed a “Levine” rat model of HI (left carotid ligation + 1 h global hypoxia on PND‐26) and used histological and electrophysiological paradigms to assess the long‐term neuroprotective properties of clomethiazole (CMZ; a GABAA receptor modulator). Key enzymes involved in inflammation, namely nitric oxide synthase (NOS) and arginase, were also examined to assess potential CMZ mechanisms not involving GABA‐R activation. Assessments were carried out 3 and 90 days post‐HI. Extensive CNS lesions were evident after HI ipsilaterally at both short‐ and long‐term intervals. CMZ significantly decreased the lesion size at 3 and 90 days (P<0.01; P<0.05). Evoked field potential analyses were used to assess hippocampal CA1 neuronal activity ex vivo. Electrophysiological measurements contralateral to the occlusion revealed impaired neuronal function after HI relative to short‐ and long‐term controls (P<0.001, 3 and 14 days; P<0.01, 90 days), with CMZ treatment providing near complete protection (P<0.001 at 3 and 14 days; P<0.01 at 90 days). Both NOS and arginase activities were significantly increased at 3 days (P<0.01), with arginase remaining elevated at 90 days post‐HI (P<0.05) ipsilaterally. CMZ suppressed the HI‐induced increase in iNOS and arginase activities (P<0.001; P<0.05). These data provide evidence of long‐term functional neuroprotection by CMZ in a model of HI. We further conclude that under conditions of HI, functional deficits are not restricted to the ipsilateral hemisphere and are due, at least in part, to changes in the activity of NOS and arginase.

Melatonin accelerates the process of wound repair in full-thickness incisional wounds

Abstract:  The pineal gland hormone melatonin is known to have both anti‐inflammatory and immunomodulatory effects. Given this, we propose that melatonin is an ideal candidate to enhance the process of wound healing. The present study assessed the effects of exogenously administered melatonin (1.2 mg/kg intra‐dermal), on scar formation using a full‐thickness incisional rat model of dermal wound healing. Melatonin treatment significantly improved the quality of scarring, both in terms of maturity and orientation of collagen fibres. An increase in nitric oxide synthase (NOS) activity and therefore nitric oxide production is detrimental during inflammation but is favourable during granulation tissue formation. Melatonin treatment significantly decreased inducible NOS (iNOS) activity during the acute inflammatory phase but significantly increased iNOS activity during the resolving phase. Cyclooxygenase‐2, which has been shown to have anti‐inflammatory effects, was elevated in the melatonin‐treated rats following wounding. In addition, melatonin treatment also accelerated the angiogenic process, increasing the formation of new blood vessels and elevating the level of vascular endothelial growth factor protein expression during granulation tissue formation. Melatonin treatment increased arginase activity (which generates proline, a building block for collagen synthesis) from earlier time points. The protein profiles of hemoxygenase‐1 (HO‐1) and HO‐2 isoforms, vital participants in the repair process, were also up‐regulated upon melatonin treatment. This study has therefore demonstrated, for the first time, that melatonin can significantly improve the quality of wound healing and scar formation.