Epicatechin gallate improves healing and reduces scar formation of incisional wounds in type 2 diabetes mellitus rat model.

Diabetic foot ulcers are the most severe clinical manifestation of diabetes-related impaired wound healing. Current standard and experimental treatments for these ulcers are largely ineffective. Epicatechin gallate (ECG) is a nontoxic flavonoid previously shown to improve normal wound healing and scar formation. In this study, the neonatal streptozotocin-induced diabetes mellitus (nSTZ-DM) type 2 model in rats was used to investigate the effects of ECG on impaired wound healing and scar formation. Administration of 100 mg/kg STZ induced a significant (P < 0.05) state of mild hyperglycemia in nSTZ-DM type 2 rats, compared to nondiabetic controls. The effects of 0.8 mg/mL ECG on wound healing were then investigated using the full-thickness incisional wound-healing model. ECG significantly improves healing and reduces scar formation in nSTZ-DM type 2 rats (P < 0.05). Biochemical improvements were also found, including significantly increased total nitric oxide synthase activity ([NOS]; P < 0.001) and inducible NOS (iNOS) activity (P < 0.01). This work highlights ECG as a potential treatment for DM-impaired wound healing. .

Melatonin treatment following stroke induction modulates L-arginine metabolism.

The efficacy of melatonin treatment in experimental stroke has been established. Some of the neuroprotective properties have been attributed to its anti-oxidant and anti-inflammatory effects. Nitric oxide synthases (NOS) and cyclooxygenases (COX) are considered to have a significant role in the inflammatory milieu occurring in acute stroke. While previous reports have shown that pretreatment with melatonin in a stroke model can modulate NOS isoforms, the effect of post-treatment with melatonin on l-arginine metabolism has not been investigated. This study initially examined the effect of melatonin (1 nm-1 mm) on l-arginine metabolism pathways in human fibrosarcoma fibroblasts (HT-1080) fibroblasts. Evidence of neuroprotection with melatonin was evaluated in rats subjected to middle cerebral artery occlusion (MCAO). Animals were treated with three daily doses of 5 mg/kg i.p., starting 1 hr after the onset of ischemia. Constitutive NOS activity but not expression was significantly increased by in vitro exposure (72 hr) to melatonin. In addition, melatonin treatment increased arginase activity by increasing arginase II expression. In vivo studies showed that melatonin treatment after MCAO significantly inhibited inducible NOS activity and attenuated expression of the inducible isoform, resulting in decreased total NOS activity and tissue nitrite levels. COX activity was significantly reduced with melatonin treatment. The neuroprotective anti-inflammatory effects of melatonin were consistent with the substantial reduction in infarct volume throughout the cortex and striatum and recovery of mitochondrial enzyme activities. The evidence presented here suggests that modulation of l-arginine metabolism by melatonin make it a valuable neuroprotective therapy for stroke.

Tin protoporphyrin provides protection following cerebral hypoxia-ischemia: involvement of alternative pathways.

The contribution of heme oxygenase (HO)-linked pathways to neurodegeneration following cerebral hypoxia-ischemia (HI) remains unclear. We investigated whether HO modulators affected HI-induced brain damage and explored potential mechanisms involved. HI was induced in 26-day-old male Wistar rats by left common carotid artery ligation, followed by exposure to a humidified atmosphere of 8% oxygen for 1 hr. Tin protoporphyrin (SnPP; an HO inhibitor), ferriprotoporphyrin (FePP; an HO inducer), or saline was administered intraperitoneally once daily from 1 day prior to HI until sacrifice at 3 days post-HI. SnPP reduced (P < 0.05) infarct volume compared with saline-treated animals, but FePP had no effect on brain injury. SnPP did not significantly inhibit HO activity at 3 days post-HI, but SnPP increased (P < 0.001) total nitric oxide synthase (NOS) activity compared with HI + saline. Both inducible NOS and cyclooxygenase activities were attenuated (P < 0.05) by SnPP, whereas mitochondrial complex I and V activities were augmented (P < 0.05) by SnPP. SnPP had no effect on NMDA receptor currents. Overall, like other HO inhibitors, SnPP produced many nonselective effects, such as attenuation of inflammatory enzymes and increased mitochondrial respiratory function, which were associated with a protective response 3 days post-HI.

Cerebral heme oxygenase 1 and 2 spatial distribution is modulated following injury from hypoxia-ischemia and middle cerebral artery occlusion in rats.

The regional and cellular distribution of heme oxygenase (HO)-1 and -2 following cerebral ischemia has not been ascertained. Employing the transient middle cerebral artery occlusion (MCAO) and hypoxia-ischemia (HI) models of unilateral brain injury, the aim was to elucidate immunolocalization of HO-1 and HO-2. Animals were sacrificed 3 days post-ischemia and immunohistochemistry and Western blotting were utilized to determine HO-1 and HO-2 expression. In the ipsilateral hemisphere following HI, HO-1 immunoreactivity was significantly upregulated in many neuronal and glial populations (including the cortex, hippocampus and thalamus). HO-1 was also detected in macrophages/microglia within the infarct. In addition to widespread neuronal HO-2 labelling, HO-2 was also expressed in vascular endothelial cells. Inflammatory cells within the infarct of MCAO and HI animals were surprisingly immunoreactive for HO-2, but only HI animals had significantly elevated HO-2 protein expression in the ipsilateral hemisphere. This may be due to the presence of global hypoxia in the HI model which can upregulate vascular endothelial growth factor and subsequent proliferation of endothelial cells. This report of HO-2 protein expression upregulation following HI coupled with an increase in HO-1 immunoreactivity suggests that this response may be implicated in reducing cell death or repairing damage induced by cerebral ischemia.

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

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.

Angiogenesis as a predictive marker of neurological outcome following hypoxia-ischemia.

Cerebral ischemia induces angiogenesis within and around infarcted tissue. The protection of existing and growth of new blood vessels may contribute to a more favorable outcome. The present study assessed whether angiogenesis can be used as a marker for neurodegeneration/neuroprotection in a model of hypoxia-ischemia (HI). Increased CD31 immunoreactivity 7 days post-HI indicated increased angiogenesis compared to controls (P<0.001). Treatment with the GABA(A) receptor modulator, clomethiazole (CMZ; 414 mg/kg/day), normalized the level of angiogenesis compared to HI + saline (P<0.001). Conversely, the non-selective nitric oxide synthase (NOS) inhibitor, L-NAME (5 mg/kg/day), markedly decreased angiogenesis compared to controls (P<0.001). Circulating plasma levels of IL-1alpha, IL-1beta and GM-CSF were significantly elevated post-HI. CMZ treatment attenuated these increases while also stimulating IL-10 levels. L-NAME treatment did not alter IL-1alpha or IL-1beta levels, but decreased endogenous IL-10 levels and exacerbated the ischemic lesion (P<0.001). CMZ treatment has been shown to increase NOS levels, while L-NAME halted the HI-induced increase in NOS activity (P<0.001). We conclude that angiogenesis can be used as a marker of neurodegeneration/neuroprotection for cerebral HI and is correlated to NOS activity and circulating inflammatory mediators.

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.

Major enzymatic pathways in dermal wound healing: current understanding and future therapeutic targets.

Skin is an essential protective organ for vertebrate animals. During skin injury, a plethora of cells and mediators occupy the wound site and, through a collective effort, perform repair of the tissue. This complex pathophysiological process is referred to as wound healing. The efficiency of wound repair is governed by the sequential influx of a variety of cell types to the wound site, upregulation/downregulation of many signaling molecules, and the interaction of various enzymatic pathways. Any dysregulation in this highly complex, but orderly, pathophysiological process results in impaired wound repair. A variety of metabolic enzymes are induced upon injury and are responsible for driving the key physiological processes within the wound milieu during the inflammatory and resolution phases of wound repair. This review will focus on the contribution of major enzymatic biosystems to the inflammatory, remodeling and resolution phases of normal wound healing, including the arachidonic acid metabolic pathway, L-arginine metabolism and the endogenous oxidant-antioxidant redox systems of the body. The major therapeutic targets within these processes will also be highlighted.

Mechanisms of action of green tea catechins, with a focus on ischemia-induced neurodegeneration.

Catechins are dietary polyphenolic compounds associated with a wide variety of beneficial health effects in vitro, in vivo and clinically. These therapeutic properties have long been attributed to the catechins' antioxidant and free radical scavenging effects. Emerging evidence has shown that catechins and their metabolites have many additional mechanisms of action by affecting numerous sites, potentiating endogenous antioxidants and eliciting dual actions during oxidative stress, ischemia and inflammation. Catechins have proven to modulate apoptosis at various points in the sequence, including altering expression of anti- and proapoptotic genes. Their anti-inflammatory effects are activated through a variety of different mechanisms, including modulation of nitric oxide synthase isoforms. Catechins' actions of attenuating oxidative stress and the inflammatory response may, in part, account for their confirmed neuroprotective capabilities following cerebral ischemia. The versatility of the mechanisms of action of catechins increases their therapeutic potential as interventions for numerous clinical disorders. However, more epidemiological and clinical studies need to be undertaken for their efficacy to be fully elucidated.

The biology and pathology of hypoxia-ischemia: an update.

After an hypoxic-ischemic (HI) insult, a multi-faceted complex cascade of events occurs that ultimately causes cell death and neurological damage to the central nervous system. The various cascades include, amongst others: immunological changes, such as the activation of the complement system and the generation of antibodies; increased inflammation through the actions of pro-inflammatory cytokines and chemokines; the production of reactive oxygen species leading to oxidative stress; and diminished mitochondrial function leading to the activation of apoptotic pathways and subsequent alteration in the function of neurons within the contralateral hemisphere. This review addresses the immunological aspects following HI, the role of various cytokines (both pro-inflammatory and anti-inflammatory) and chemokines after the induction of HI. In addition, the role of free radicals in producing HI-induced neurodegeneration and the contribution that mitochondrial dysfunction has in neuronal apoptotic cell death will be discussed. This review also covers the changes that the previously assumed "internal control", the contralateral hemisphere, undergoes due to HI and describes the difficulties associated with therapy intended to prevent neuronal injury associated with HI.

(-)-Epigallocatechin gallate as an intervention for the acute treatment of cerebral ischemia.

This study examined the neuroprotective effects and possible hepatotoxicity of (-)-epigallocatechin gallate (EGCG) in a rat model of transient focal cerebral ischemia. Male Sprague-Dawley rats (265-295 g) were treated with either 50 mg kg(-1) of EGCG or saline, i.p., immediately post-ischemia and every day thereafter, in a middle cerebral artery occlusion model of stroke. Sacrifice occurred 72 h post-ischemia and 2,3,5-triphenyltetrazolium chloride staining was used to quantify neuronal infarction. Hepatotoxicity was determined by taking blood samples for plasma alanine aminotransferase (ALT) activity. Spleen, kidney, liver and testes wet weights were also recorded. Total infarct volume was significantly (P<0.05) reduced in the EGCG-treated group as compared to controls. Analysis of the mean infarct area showed a significant (P<0.05) decrease in slices 6 and 7 in the EGCG-treated group. No significant differences were found in organ weights or ALT levels between treatment groups. Our findings, in part, validate and extend previous observations illustrating that 50 mg kg(-1), i.p. EGCG is non-toxic and neuroprotective. However, we also found that EGCG treatment appreciably increased (>50%) the number of animals that developed an intracerebral hemorrhage. We therefore conclude that 50 mg kg(-1) EGCG is not a viable intervention for the acute treatment of cerebral ischemia, as it is likely to increase the risk of intracerebral hemorrhaging.

Hippocampal nitric oxide synthase and arginase and age-associated behavioral deficits.

The present study investigated age-related changes in nitric oxide synthase (NOS) and arginase in the subregions of the hippocampus and their correlations with animals' performance in the open field, T-maze, and water maze tasks. Aged rats (24 months old) showed reduced exploratory activity and poorer spatial learning relative to the young adults (4 months old). Significant increases in total NOS activity were found in the aged dentate gyrus and a dramatic decrease in endothelial NOS expression was observed in the aged CA2/3. Activity or protein expression of inducible NOS was not detected in any subregion of the hippocampus. There were no age-related changes in total arginase activity or arginase I and arginase II protein expression. Correlation analysis revealed that animals' motor ability was associated with CA1 NOS and arginase, as well as hippocampal function. The present findings provide further support for the involvement of NOS/NO and arginase in the normal aging process. A strong positive correlation between CA1 eNOS protein expression and swimming speed in the water maze task may reflect a relationship between the local cerebral blood flow and neuronal activity.

Possible anti-inflammatory role of COX-2-derived prostaglandins: implications for inflammation research.

Inflammation is the response of any tissue to injury or trauma. The inflammatory response forms the basis of several pathological and pathophysiological processes, including wound healing, rheumatoid arthritis and neurodegenerative disorders, including Alzheimer's disease and stroke. The response is mediated by various signaling molecules and enzymatic pathways, among which the cyclooxygenase (COX) pathway is one of the most predominant. COX catalyzes the formation of prostaglandins and thromboxanes from arachidonic acid (AA). In 1971, Sir John Vane demonstrated for the first time that the mechanism of action of aspirin and other non-steroidal anti-inflammatory drugs (NSAIDs) is via inhibition of COX. Since the discovery of at least two isoforms of COX, inhibition of COX-2 has generally been considered the basis for the anti-inflammatory effects of NSAIDs. However, more recent studies of COX-2 have controversially postulated that the selective inhibition of COX-2 may not be beneficial, but rather can be detrimental. This is based on the observations that, during inflammation, not all AA-derived mediators are exclusively pro-inflammatory. This review will attempt to discuss the confusing and contradictory data relating to selective COX-2 inhibition and inflammation, particularly focusing on anti-inflammatory AA-derived mediators. We will also try to advance the perspective that inflammation is not just a single process, but is rather a dynamic and continuously changing event, not just in terms of time, mediators and cells, but also in the initiating stimuli, whether it be in the periphery or the central nervous system.

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 GABA(A) 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.

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 mm(3)) in comparison to HI + saline (99.6+/-15.6 mm(3)). In addition, EGCG significantly reduced total (622.6+/-85.8 pmol L-[(3)H]citrulline/30 min/mg protein) and inducible nitric oxide synthase (iNOS) activity (143.2+/-77.3 pmol L-[(3)H]citrulline/30 min/mg protein) in comparison to HI+saline controls (996.6+/-113.6 and 329.7+/-59.6 pmol L-[(3)H]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.

Immunohistochemical localization of cerebrovascular cannabinoid CB1 receptor protein.

Cannabinoids are powerful hypotensives and vasodilators. However, their mode of action is controversial. This study is the first to investigate the distribution of vascular CB1 receptor protein expression in situ. We used double-fluorescence and chromogenic immunohistochemistry to investigate patterns of CB1 protein expression in cerebrovascular tissue in rat brain sections. We found a layer of intense CB1 labeling immediately adjacent to the internal elastic lamina, consistent with myointimal and vascular smooth muscle cells, and diffuse labeling adventitial to this layer. We concluded that CB1 receptor are most intensely expressed in the vascular smooth muscle layer in cerebral arteries, and are likely to be chiefly responsible for the potent vasodilatory effect of cannabinoids.

Inflammation and autoimmunity as a central theme in neurodegenerative disorders: fact or fiction?

Irrespective of the initiating stimuli, neurodegenerative disorders including multiple sclerosis (MS), Alzheimer's disease, Parkinson's disease and stroke share many characteristics of inflammation and autoimmunity. This review summarizes and correlates the information relating to the role of cytokines and chemokines in initiating and propagating the inflammatory/immune response in these pathologies. For example, in MS there is a continuous realignment in the inflammatory and immune response. However, due to the redundancy in the cytokine/chemokine response, it is extremely unlikely that any one therapy will be successful in treating neurodegenerative diseases. This review attempts to highlight specific targets for therapeutic intervention.

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.

Cannabinoid CB1 receptor protein expression in the rat choroid plexus: a possible involvement of cannabinoids in the regulation of cerebrospinal fluid.

Cannabinoid CB1 receptors in the brain are expressed on axon terminals presynaptic to neurons that express fatty acid amide hydrolase (FAAH). Postsynaptic FAAH catabolizes endocannabinoids which act as short-range transmitters. It has been previously shown that FAAH is also expressed in the epithelial cells of the choroid plexus. Using immunohistochemisty, we found that CB1 receptor protein is also expressed in choroid plexus epithelia. This is consistent with the hypothesis that FAAH in choroid plexus epithelial cells catabolizes endocannabinoids close to their site of action. Cannabinoids may then act directly on choroid plexus cells, and thereby contribute to the regulation of the composition of the CSF.

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 +/- 10.4 mm3 (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.