Purified herba leonuri and leonurine protect middle cerebral artery occluded rats from brain injury through antioxidative mechanism and mitochondrial protection 5

From this study, it is showed that DNA oxidative damage was increased after MCAO, suggesting the increase free radical activity, as well as reciprocal decreased endogenous antioxidant ac

Chapter 5 Discussion

During ischemia, the disruption of cerebral blood flow deprives brain cells of oxygen, leading to a reduction in energy production associated with building-up toxic metabolites such as glutamate, inflammatory mediators, free radicals that could ultimately trigger ischemic cascade As mentioned in section 2.1.3, over production of free radicals during ischemia results in oxidative stress Several possible mechanisms have been postulated for the generation of ROS during ischemia (Loh et al, 2006a) The inherent biochemical and physiological characteristics of brain, including high poly-unsaturated fatty acid and the energy requirements, make it particularly vulnerable to the oxidative stress Oxidative stress does not play in isolation, but participates in the complex interplay between excitotoxicity, apoptosis, inflammation and so forth Low antioxidant defense mechanisms and high oxygen consumption again subjects the brain to be more vulnerable

to the oxidative stress insult Therefore, modulation of oxidative stress could be a potential therapeutic approach for various neurodegenerative diseases

Mitochondria are the primary cellular source of ROS as they generate huge numbers of oxidation-reduction reactions and they consume massive amounts of oxygen Mitochondrial dysfunction is also well-documented in the ischemic cascade Thus, mitochondria are both the initiator and target of oxidative stress Mitochondria damage leads to cell death given the role of mitochondria in the energy metabolism, calcium homeostasis and programmed cell death Therefore, prevention of mitochondrial damage could confer a protective mechanism to ischemic stroke

of motor function as measured by neurological deficit grading system Under the treatment of pHL, the infarct volume was significantly reduced This phenomenon indicates the lesser histological damage, possibly within the area of penumbra was resulted in the stroke group treated with pHL as simplistic view of ischemic penumbra suggests cell death in this area undergo apoptosis which could be possibly inhibited but not necrosis cell death in core infarct Neurological impairment was slightly alleviated by pHL 1 day after MCAO Significant difference of functional outcome was observed between vehicle group and stroke group treated with pHL 7 days after MCAO, as measured by neurological deficit grading system As ample evidences from 15O (Furlan et

al, 1996; Heiss et al, 1998) and 18F-fluoromisonidazole (Read et al, 2000; Markus et al, 2004) PET studies indicate that survival of the tissue in penumbra has benefit on subsequent neurological recovery A positive correlation does exist with improvement of

2 month recovery scores and the survival of penumbra, leading to the hypothesis that survival of penumbra influences late recovery, possibly through allowing the subsequent

peri-infarct neuronal reorganization (Moustafa and Baron, 2008) With this rationale, pHL is suggested to have the effect of preventing the damage within the affected area, particularly through the protection of ischemic penumbra

Oxidative stress is associated with MCAO as shown by reduced plasma total antioxidant concentration and enhanced cerebral DNA oxidative damage pHL treatment was shown to ameliorate oxidative stress in MCAO-induced rats

Endogenous antioxidant system under the influence of pHL was also investigated Plasma was selected as the sample for this experiment as it contains many non-enzymatic antioxidants and antioxidant enzymes The major antioxidant defenses in plasma include ascorbate, protein thiols, bilirubin, urate and α-tocopherol (Nicholas, 1993) Plasma contains also preventive antioxidants such as caeruloplasmin and transferring Ironscavenging proteins present in plasma can contribute to the total antioxidant capacity because they scavenge the iron availability (Stocks, 1974) The total antioxidant concentration decreased significantly after MCAO This result is in consistent with the work done previously by Antonio et al (2000), which showed that mean plasma levels of nonenzymatic antioxidants and antioxidant enzymes activities were lower in patients than

in healthy controls (Antonio, 2000) Sun et al (2005) also showed that rat undergone myocardial infarction had lower level of antioxidant enzyme activities in hearts (Sun et al, 2005) The low level of antioxidant capacity in stroke groups suggests that much of the antioxidant molecules have been used up and react with ROS that over produced during ischemia Under the pHL treatment, both in healthy or stroke group, the plasma

antioxidant concentration was greatly enhanced This implies that pHL may have enhancing effects on endogenous antioxidants with or without oxidative stress condition,

thereby confer protective barrier against stroke

Single strand break and base modifications characteristic of oxidative injury to DNA can

be detected after permanent brain ischemia Free radicals cause also the DNA strand break with a less extent by nitric oxide Nitric oxide is capable of causing base modifications such as nitration or deamination (Love S, 1999) With many base modifications produced, the major base modifications that result from the reaction between peroxynitrite and DNA are the conversion of guanine to form 8-nitroguanine and the deamination of guanine to form xanthine (Love S, 1999)

Level of DNA oxidative damage was elevaluated as one of the marker of oxidative stress GC/MS has of late been widely used to quantify DNA damage and the level of oxidative stress due to its ability to identify a wide range of DNA base product in a single analysis For the DNA oxidative damage analysis, direct damage by ROS that can affect purine, pyrimidine bases and/or deoxyribose sugar was focused (Halliwell, 4th Edition) From this study, it is showed that DNA oxidative damage was increased after MCAO, suggesting the increase free radical activity, as well as reciprocal decreased endogenous antioxidant activity contributes to DNA oxidative damage after stroke insult Stroke rats treated with pHL not only had high endogenous antioxidant concentration but also had significant

reduction on DNA oxidative damage Therefore, it is proposed that the in vivo therapeutic

effects of pHL might be strongly related to its antioxidant effects by enhancing the endogenous antioxidant capacity and also alleviating the oxidative stress

TUNEL assay and Immunohistochemical staining of apoptotic-related proteins showing the presence of these proteins only in the infarct area after MCAO indicates the apoptosis involvement during ischemia injury Significant reduction of green fluorescence and immunoreactivity of pro-apoptotic proteins could be observed in stroke rats with pHL treatment

Experimental animal models of global ischemia and focal ischemia have suggested the involvement of cell survival/death signaling pathway in the pathogenesis of cerebral ischemia (Sugawara et al, 2004) Morphological and biochemical evidences of apoptosis have also well documented in experimental animal models of cerebral ischemic injury (Charriaut-Marlangeu et al, 1996; Li et al, 1997; Fujimura et al, 1998) It is clear that both necrosis and apoptosis contribute to the cerebral ischemic injury, the relative contribution of each form varies substantially with the experimental model, brain cell type, and age of the experimental animal (Starkov et al, 2004)

A simplistic view introduced in chapter 2, in focal ischemia, there is rarely complete blockade of blood flow to the affected region because plethora of collateral vessels provides some flow to the area, making the affected region a core infarct area and an ischemic penumbra The cells in the core infarct are destined to die by necrosis while those in ischemic penumbra still receive collateral flow that sufficient to prevent cell

death by necrosis, but apoptosis There is indeed a clear indication of initiation of apoptosis pathway in the core infarct, such as cytoplasmic and nuclear condensations and activation of specific caspase cascade (Benchoua et al, 2001) However, complete morphological changes of apoptosis are not observed at the end stages of infarction The abortion of apoptosis in the core infarct might be attributed by the severe impairment of energy level that might cause a shift towards secondary necrosis from apoptosis (Nicotera

et al, 1998) In contrast, many studies have demonstrated that cells in ischemic penumbra undergo delayed cell death by apoptosis due to the allowance given by the residual blood supply to maintain a low level of ATP On the other hand, activated calpains and caspases could eventually cleave and inactivate the ion pumps This situation could lead

to the disruption of ion homeostasis and switch the apoptosis signaling to necrosis (Schwab et al, 2002) Therefore, the cell death in the ischemic area is rather a complex phenomenon Since the necrosis is beyond the therapeutic reach, the rational treatment of stroke has likewise focused on protecting the cells from undergoing apoptosis at the early stage of stroke pathology

In past, direct treatment of oxidants like hydrogen peroxide was thought to induce exclusively necrosis However, more recent studies have shown that lower doses of free radicals can trigger apoptosis as well Reciprocally, broad-spectrum of anti-apoptotic proteins such as BCL-2 have been ascribed as an antioxidant function by maintaining cells in a more reduced state by scavenging ROS either directly or by up-regulating other ROS scavengers such as thiol compounds and antioxidant enzymes, further indicating that ROS generation is one of the crucial initiation step for apoptotic event (Chandra et al,

2000) Recently, it has also been shown that oxidative stress can lead to apoptosis by inducing mitochondria permeability transition pore (MPTP) opening (Galindo et al, 2003) In addition, ROS can also lead to oxidation of the anionic phospholipids

cardiolipin to facilitate cytochrome c release from mitochondria (Halliwell, 4th Edition)

Since ample evidences demonstrate the link between oxidative stress and apoptosis, we would like to investigate whether pHL with antioxidant properties ameliorates the level

of apoptosis during stroke The TUNEL system is designed for detection of nuclear DNA fragmentation, an important biochemical hallmark of apoptosis in many cell types It has been suggested that caspase 3 may be the key executioner of nuclear degradation in ischemic neuron and contributes to the DNA fragmentation, possibly by activation of caspase-activated deoxyribonuclease (CAD) (Nakka et al, 2008) Results obtained from TUNEL assay showed that green fluorescence could only detect in infarct zone, indicating that apoptosis occurs only at the affected region In addition, strongest nuclear green fluorescence was observed in infarct zone of stroke-induced rats, which is correlated with the results obtained from other analysis in this study Lesser nuclear green fluorescence was observed in the stroke rats treated with pHL Together with its ability of reducing infarct volume, neurological impairment and DNA oxidative damage, result obtained from TUNEL assay further indicates the therapeutic potential of pHL in the

middle cerebral artery occluded Wistar rats, most probably through its antioxidant and

anti-apoptosis properties

Although TUNEL assay has been widely accepted for the detection of apoptosis, it is vulnerable to false-positives in the form of non-apoptotic necrotic cells (Iijima T, 2006) Therefore, it is always recommended to confirm the diagnosis with additional techniques such as study based on the criteria of morphological changes of apoptosis In this experiment, existence of apoptosis was confirmed through immunohistochemical staining via probing the apoptotic-related proteins

Generally, apoptosis can be classified as intrinsic pathway and extrinsic pathway There are considerable cross talks between both pathways Subsequently, they converge to result ultimately in cellular morphological and biochemical alterations characteristic of apoptosis (Ashe et al, 2003) For Fas, it is involved in the extrinsic pathway of apoptosis (Ashe et al, 2003) In healthy individuals, Fas ligand (FasL) and Fas are mainly restricted

to the immuno tissues and to sites of immune privilege such as the eye and the testes where they can trigger apoptosis of invading immune cells The function of this death inducing receptor in the healthy brain is not so clear though it was suggested that it has a role of maintenance of immune privilege through FasL binding in brain (Mehmet, 2001)

In particular, it is well documented that up-regulation of Fas is involved in ischemic injury in adult brain, which has also been shown in this experiment According to the Mehmet’s report (2001), Fas knockout mice have a reduction of 54% of infarct area when the mice were subjected to MCAO, indicating the important role of Fas in pathophysiological progression of stroke

Central to intrinsic pathway are proteins in the Bcl-2 family The Bcl-2 family can be subdivided into pro- and anti-apoptotic proteins Bax belongs to the pro-apoptotic protein while Bcl-2 and Bcl-xL belong to anti-apoptotic proteins (Love, 2003) Both Bcl-2 and Bcl-xL are localized at the mitochondrial outer membrane and to the endoplasmic reticulum and perinuclear membrane (Nakka et al, 2008) Bcl-2 is expressed at high level during the development and down-regulated after birth in the CNS In peripheral nervous system, Bcl-2 expression is maintained throughout the life and its deletion causes profound loss of peripheral motor, sensory and sympathetic neurons after birth (Merry and Korsmeyer, 1997) Bcl-xL is expressed throughout life as well (Gonzalez-Garcia et al, 1995), indicating crucial role of Bcl-2 and Bcl-xL in neuronal survival Bcl-2 and Bcl-xL could also inhibit pro-apoptotic Bcl-2 family through heterodimerization such as Bax (Nakka et al, 2008) Furthermore, Bcl-2 inhibits apoptosis through increasing mitochondrial redox capacity and increase resistance for the formation of mitochondrial transition permeability pore opening caused by the Ca2+and oxidative stress (Starkov et

al, 2004)

Consistent with the previous works done (Ferrer et al, 2003; Sairanen et al, 2001), immunohistochemical staining results showed the presence of pro-apoptotic proteins BAX and FAS only in the infarct area after MCAO but undetectable in the healthy individuals Highest immunoreactivity of pro-apoptotic proteins in vehicle group is correlated with observation of strongest nuclear green fluorescence in vehicle This indicates the up-regulation of pro-apoptotic proteins and thereby intense cell death with dramatic morphological changes via both intrinsic (BAX upregulation) and extrinsic

pathways (FAS upregulation) occurs in the region of infarct area In contrast, antiapoptotic proteins BCL-XL and BCL-2 were found in entire brain section in both sham and sham with pHL group Weaker signal could also be observed in vehicle group Significant reduction of immunoreactivity of pro-apoptotic proteins and enhancement of anti-apoptotic protein signals could be observed in stroke rats with drug treatments, suggesting the protective effects against neuronal loss within ischemic margin, after experimental stroke Zhao et al (2003) demonstrated that protection by overexpression of Bcl-2 is accompanied by reduced cytochrome c translocation to the cytosol and reduced activation of caspase 3 This observation leads us to conclude that pHL with antioxidant property has effect on apoptosis by switching the cells towards more anti-apoptotic state

In conclusion, oxidative stress process is a complex interplay that involves in cellular damage and cell death Therefore, studies focusing on antioxidant properties of Chinese herbs have been shown its promising therapeutic effects against stroke In experiment I, pHL has shown its therapeutic potential on reducing infarct volume, neurological impairment, oxidative damage and apoptosis, probably through its antioxidant effect, at least through the ability of increasing the endogenous antioxidant capacity Therefore, pharmacological modification of oxidative damage is one of the most promising avenues

5.2 Discussion on experiment II

Differential centrifugation was employed for mitochondrial preparation JC-1 assay showed that isolated mitochondria are suitable for bioenergetics studies and the isolated mitochondria are more responsive to succinate

Differential centrifugation and purification by Percoll density gradient was employed for the cortical mitochondria preparation from rats Fatty acid free bovine serum albumin (BSA) was added during the mitochondrial isolation procedures to ensure the isolated mitochondria were well-coupled, as to avoid mitochondrial uncoupling due to contaminating fatty acids (Casley et al, 2002)

At low membrane potential, JC-1 exists as green monomer in the cytosol With the increase of mitochondrial membrane potential (eg depolarization), the negative charge established by intact mitochondrial membrane potential allows the JC-1, a lipophilic dye, bearing a delocalized positive charge, to enter and aggregates in the mitochondrial matrix

as red fluorescence (Cossarizza et al, 1993) JC-1 assay showed that mitochondria prepared from rat brain contained intact mitochondria suitable for studies of bioenergetics The increase of green fluorescence of J monomer without exogenous substrates, with less intense increase of red fluorescence of J aggregates indicates that mitochondrial uptake of JC-1 as green monomers was apparently the basal conditions Upon subsequent addition

of mitochondrial substrates which oxidation energized the mitochondria, intense uptake

of JC-1 as red fluorescence of J aggregates was observed, indicating an increase of mitochondrial membrane potential The situations were reversed by adding the inhibitors

of mitochondrial substrates, indicating that isolated mitochondria were well-coupled Succinate was selected as substrate to energize the mitochondria for the subsequent experiments as from JC-1 assay, it showed that mitochondria had a larger and better response to succinate than complex I substrate (malate/glutamate)

Mitochondria are the site of ROS generation, and mitochondrial ROS generation was reduced by pHL treatment under both physiological and H 2 O 2 -treated condition

Under physiological condition, 2% of electrons passing through the mitochondrial ETC leak out and react with molecular oxygen to form superoxide anions (Christophe and Nicolas, 2006) It is unknown that the mitochondrial redox sites responsible for the ROS

generation in vivo Experiments in vitro demonstrated that multiple redox sites in the

ETC responsible for the ROS generation with the differences in their efficacy and mechanisms of generation In the presence of complex III inhibitor antimycin A, complex III generates a substantial amount of superoxide However, contribution of complex III for ROS generation in the absence of inhibitor remains to be resolved (Starkov et al, 2004) Another site of ROS generation is located in the complex I in respiration chain It was demonstrated that ROS production at complex I is supported by the oxidation of NAD-reducing substrates which is stimulated by both high membrane potential and reduced redox state of intramitochondrial NAD(P)H (Starkov and Fiskum, 2003)

Under physiological conditions, O2˙¯ is rapidly dismutated by superoxide dismutase (Mn-SOD) to H2O2 Owing to the longer half time and greater liposolubility, H2O2 is

more readily diffused out from the mitochondria It is then subsequently converted to water by glutathione peroxidase (GPx) or catalase In addition to these antioxidant enzymes, mitochondria may limit the ROS generation via glutathione, ascorbic acid and alpha-tocopherol (vitamin E) (Christophe and Nicolas, 2006) Therefore, high efficient scavenging systems within the healthy mitochondria suggest that any measured release of ROS from mitochondria may represent only a small fraction of the total ROS generated

Since mitochondria have a key role in ROS production, the effect of pHL on the mitochondrial ROS production was firstly determined DCFDA is a nonfluorescent probe, which upon oxidation, mainly by peroxides, is converted to the highly fluorescent derivatives DCF The obtained data showed that addition of succinate into mitochondrial suspension caused the increased of endogenous generation of ROS ROS generation stimulated by succinate oxidation via reverse electron flow from complex II and complex

I requires a high membrane potential and a highly reduced redox state of ubiquinone The source of reducing power is from FADH2-dependent substrates such as succinate or α- glycerophosphate (Starkov and Fiskum, 2003) The physiological concentration of succinate is relatively under normal condition (0.2-0.4mM) However, it increased dramatically (up to 4-7mM) during brain ischemia This accumulation of succinate during brain ischemia provides the condition of high mitochondrial membrane potential and reducing power necessary for the reverse flow of electron from complex II to complex I (Starkov et al, 2004) Similar result was reported by Starkov and Fiskum (2003) that the rate of H2O2 production increased robustly after addition of respiratory substrate α-

ketoglutarate and was closely proportional to the increased mitochondrial membrane potential and the increased level of NAD(P)H

To mimic the condition of oxidative stress, mitochondria were treated with H2O2 in certain groups of experiments Although H2O2 is not strictly a ROS as it lacks unpaired electron, it is a potent oxidant and freely diffuse across the mitochondrial membrane In mitochondria, H2O2 can be metabolized by GSH in mitochondria or converted to hydroxyl radical via Haber-Weiss and Fenton reactions (Christophe and Nicolas, 2006) With the treatment of H2O2, mitochondrial ROS generation was greatly enhanced as compared to the group energized by succinate alone, indicating H2O2 has also the potential to be converted to other damaging radicals to cause free radical toxicity For the effect of drug, pHL has effect on reduction of mitochondrial ROS generation in both physiological and H2O2-treated conditions, suggesting that administration of antioxidants might confer a protective effect during ischemic insult by reducing mitochondrial ROS generation

Reduced ATP biosynthesis was observed in H 2 O 2 -treated mitochondria ATP biosynthesis was suppressed by pHL treatment, which is hypothesized be the effect of metabolic arrest to mitochondria

Under physiological condition as mitochondria is normally energized, complex V (F0F1- ATP synthase) synthesizes ATP from ADP and phosphate In contrast, during ischemia, decrease in oxidative phosphorylation induces a drop in ATP production, and increases

the level of ADP and AMP This is due to the reverse reaction of F0F1-ATPase in this case, that hydrolyses the ATP to ADP and phosphate, and becomes the major consumer

of ATP during ischemia Large consumption of ATP by mitochondria during ischemia is believed to be involved in the maintenance of mitochondrial membrane potential

However, the drop in ATP will subsequently favor the entry of Ca2+, generation of ROS which could still ultimately cause mitochondrial damage (Christophe and Nicolas, 2006)

It was reported that generation of ROS under this condition is driven by mitochondrial hyperpolarization (high membrane potential), which is initiated by the inhibition of

reentry of protons via F0F1-ATPase during brain ischemia (Iijima T, 2006) In agreement with the hypothesis, it is founded that mitochondrial ATP biosynthesis was greatly

suppressed upon challenged by H2O2, suggesting that reduction in mitochondrial ATP

biosynthesis upon treatment of H2O2 was accompanied with huge increase in ROS generation and it was largely due to the reverse reaction of complex V

Under both physiological and H2O2 treated conditions, pHL suppressed the mitochondrial ATP biosynthesis It is believed that pHL acts differently from H2O2 in suppressing ATP biosynthesis pHL might have effect of metabolic arrest to mitochondria Scientists describe this phenomenon of drastic reduction in energy production (metabolism) and energy consumption (cellular activity) as quiescence, torpor or hibernation in terms of different extent of suspended animation With more, organisms in this state confer a greater resistance to environmental stress such as temperature extremes, oxygen deprivation or physical injury (Roth and Nystul, 2005)

According to Geng and Su (1991), Chinese herbs are generally classified into four categories: “Yang-invigorating”, “Yin-invigorating”, “Qi-invigorating” and “Blood-enriching”, with “Qi-invigorating” and “Blood-enriching” belong to Yang and Yin family While maintaining the balance of Yang and Yin is akin to attaining the homeostasis in the modern medicine (Ko et al, 2006) Interesting work done by Ko and co-workers (2006) showed that Yang-invigorating herbs promotes mitochondrial electron transport chain and promote ATP biosynthesis None of the Yin-invigorating herbs exhibited enhancing effect of ATP biosynthesis and most of the “Qi-invigorating” herbs suppressed ATP biosynthesis In addition, “Qi-invigorating” herbs stimulate ETC activity regardless of whether they promote or suppress ATP biosynthesis They hypothesize that the decrease

of ATP generating capacity by most of the “Qi-invigorating” herbs reflected a dynamic process involving the generation and consumption of ATP whereby ATP-consuming activities overwhelm those of ATP generation for the case of “Qi-invigorating” herbs (Ko

et al, 2006) By referring to their work, we suggest and categorize the pHL might be a

“Qi-invigorating” herb which has the effect of suppressing mitochondrial ATP biosynthesis and increase in ETC as shown by the increase of mitochondrial respiration (oxygen consumption)

Slight increase of state 4 respiration in isolated mitochondria and increase of state 3 respiration in vivo were observed under pHL treatment

According to the in vitro study, although metabolic rate (mitochondrial respiration) did

not change significantly with the pHL treatment in isolated mitochondria, a slight

decrease of RCR by increase of state 4 respiration was observed by pHL treatment in a dose-dependent manner (Figure 4-11) This phenomenon prompts us to suggest that pHL

might have a mild uncoupling function in vitro that collapse the proton gradient, thereby

decreasing the protonmotive force across the inner membrane The uncoupling effect

“uncouples” the pumping of protons out of the matrix via ETC from the flow of protons through the ATP synthase While it is obvious that long term and complete uncoupling of mitochondrial could be deleterious, mild or transient uncoupling effect has first been reported by Skulachev (1996) that it could be beneficial and it is now believed to be neuroprotective (Sullivan et al, 2004) Uncoupling effect would facilitate the increased rates of electron transfer to the molecular oxygen, with coincidental reduction in the mitochondrial membrane potential, thereby preventing any back-up of electrons in the ETC Since the magnitude of ROS production is largely dependent on the membrane potential that a modest reduction via increased proton conduction across the mitochondrial inner membrane (uncoupling) reduces the ROS formation, this could reduce the production of ROS and therefore ameliorate the ROS-induced oxidative damage (Boutilier and St-Pierre, 2002)

Uncoupling effect is achieved by facilitating the movement of protons from the mitochondrial inner membrane space into the mitochondrial matrix (Sallivan et al, 2004)

In addition, uncoupling effect may occur via changes in membrane structure that lead to proton leakage, or via an increase in the activity of inner mitochondrial membrane proteins, called uncoupling proteins (UCP), which mediate the fast transfer of fatty acid anion across the inner mitochondrial membrane to dissipate the electrochemical proton

gradient (Skulachev VP, 1997) Diminished brain damage resulted from experimental stroke and traumatic brain injury was observed in mice over-expressing human UCP-2 (Mattiasson et al, 2003) UCP-2 is suggested to play a role in neuroprotection by inhibiting ROS production by mitochondria, inhibiting ATP synthesis by complex V, limit the mitochondrial Ca2+-buffering capacity so that to prevent the detrimental effects from Ca2+ overload Most importantly, UCP-2 lowers the mitochondrial membrane potential and uncouples oxygen consumption by ETC It dissipates the proton-electrochemical gradient, thereby improving the efficiency of electron transfer between complex I and IV, and reducing the electron leakage through complex I and III (Christophe and Nicholas, 2006) Therefore, the identification of exogenous compounds that can activate UCP2 would be a step forward along this avenue of research

In addition to UCP-2, mitochondrial ATP-sensitive K+ channel (mitoKATP) can also be one of the candidates that pHL acting on Brain mitochondria exhibit the highest levels of mitoKATP proteins and K+ transport activity in any other mammalian tissues (Bajgar et al, 2001) Therefore, any possible effect of mitoKATP on ROS generation will be intensified

in the brain This channel promotes K+ entrance into the mitochondrial matrix in a manner stimulated by membrane potential Enhanced K+ cycling through mitoKATP leads

to increased mitochondrial volume, faster respiration, lower membrane potential, increased mitochondrial pH with matrix alkalinization and reduction in ROS production (Ferranti et al, 2003) It does not affect mitochondrial function as the K+ transport through mitoKATP is limited due to the low quantity and transport rates of the channel MitoKATP

decreased ROS generation is the presence of absence of oxidative phosphorylation, and