Intranasal delivery of mitochondria targeted neuroprotective compounds for traumatic brain injury: screening based on pharmacological and physiological properties.

Targeting drugs to the mitochondrial level shows great promise for acute and chronic treatment of traumatic brain injury (TBI) in both military and civilian sectors. Perhaps the greatest obstacle to the successful delivery of drug therapies is the blood brain barrier (BBB). Intracerebroventricular and intraparenchymal routes may provide effective delivery of small and large molecule therapies for preclinical neuroprotection studies. However, clinically these delivery methods are invasive, and risk inadequate exposure to injured brain regions due to the rapid turnover of cerebral spinal fluid. The direct intranasal drug delivery approach to therapeutics holds great promise for the treatment of central nervous system (CNS) disorders, as this route is non-invasive, bypasses the BBB, enhances the bioavailability, facilitates drug dose reduction, and reduces adverse systemic effects. Using the intranasal method in animal models, researchers have successfully reduced stroke damage, reversed Alzheimer's neurodegeneration, reduced anxiety, improved memory, and delivered neurotrophic factors and neural stem cells to the brain. Based on literature spanning the past several decades, this review aims to highlight the advantages of intranasal administration over conventional routes for TBI, and other CNS disorders. More specifically, we have identified and compiled a list of most relevant mitochondria-targeted neuroprotective compounds for intranasal administration based on their mechanisms of action and pharmacological properties. Further, this review also discusses key considerations when selecting and testing future mitochondria-targeted drugs given intranasally for TBI.

Revert total protein normalization method offers a reliable loading control for mitochondrial samples following TBI.

Owing to evidence that mitochondrial dysfunction plays a dominant role in the traumatic brain injury (TBI) pathophysiology, the Western blot (WB) based immunoblotting method is widely employed to identify changes in the mitochondrial protein expressions after neurotrauma. In WB method, the housekeeping proteins (HKPs) expression is routinely used as an internal control for sample normalization. However, the traditionally employed HKPs can be susceptible to complex cascades of TBI pathogenesis, leading to their inconsistent expression. Remarkably, our data illustrated here that mitochondrial HKPs, including Voltage-dependent anion channels (VDAC), Complex-IV, Cytochrome C and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) yielded altered expressions following penetrating TBI (PTBI) as compared to Sham. Therefore, our goal was to identify more precise normalization procedure in WB. Adult male Sprague Dawley rats (N = 6 rats/group) were used to perform PTBI, and the novel REVERT Total Protein (RTP) method was used to quantify mitochondrial protein load consistency between samples at 6 h and 24 h post-injury. Notably, the RTP method displayed superior protein normalization compared to HKPs method with higher sensitivity at both time-points between experimental groups. Our data favors application of RTP based normalization to accurately quantify protein expression where inconsistent HKPs may be evident in neuroscience research.

Early Thalamocortical Reperfusion Leads to Neurologic Recovery in a Rodent Cardiac Arrest Model.

BACKGROUND: Cerebral blood flow (CBF) plays an important role in neurological recovery after cardiac arrest (CA) resuscitation. However, the variations of CBF recovery in distinct brain regions and its correlation with neurologic recovery after return of spontaneous circulation (ROSC) have not been characterized. This study aimed to investigate the characteristics of regional cerebral reperfusion following resuscitation in predicting neurological recovery. METHODS: Twelve adult male Wistar rats were studied, ten resuscitated from 7-min asphyxial CA and two uninjured rats, which were designated as healthy controls (HCs). Dynamic changes in CBF in the cerebral cortex, hippocampus, thalamus, brainstem, and cerebellum were assessed by pseudocontinuous arterial spin labeling magnetic resonance imaging, starting at 60 min after ROSC to 156 min (or time to spontaneous arousal). Neurologic outcomes were evaluated by the neurologic deficit scale at 24 h post-ROSC in a blinded manner. Correlations between regional CBF (rCBF) and neurological recovery were undertaken. RESULTS: All post-CA animals were found to be nonresponsive during the 60-156 min post ROSC, with reductions in rCBF by 24-42% compared with HC. Analyses of rCBF during the post-ROSC time window from 60 to 156 min showed the rCBF recovery of hippocampus and thalamus were positively associated with better neurological outcomes (rs = 0.82, p = 0.004 and rs = 0.73, p < 0.001, respectively). During 96 min before arousal, thalamic and cortical rCBF exhibited positive correlations with neurological recovery (rs = 0.80, p < 0.001 and rs = 0.65, p < 0.001, respectively); for predicting a favorable neurological outcome, the thalamic rCBF threshold was above 50.84 ml/100 g/min (34% of HC) (area under the curve of 0.96), whereas the cortical rCBF threshold was above 60.43 ml/100 g/min (38% of HC) (area under the curve of 0.88). CONCLUSIONS: Early magnetic resonance imaging analyses showed early rCBF recovery in thalamus, hippocampus, and cortex post ROSC was positively correlated with neurological outcomes at 24 h. Our findings suggest new translational insights into the regional reperfusion and the time window that may be critical in neurological recovery and warrant further validation.

Acute-stage MRI cerebral oxygen consumption biomarkers predict 24-hour neurological outcome in a rat cardiac arrest model.

Brain injury following cardiac arrest (CA) is thought to be caused by a sudden loss of blood flow resulting in disruption in oxygen delivery, neural function and metabolism. However, temporal trajectories of the brain's physiology in the first few hours following CA have not been fully characterized. Furthermore, the extent to which these early measures can predict future neurological outcomes has not been determined. The present study sought to perform dynamic measurements of cerebral blood flow (CBF), oxygen extraction fraction (OEF) and cerebral metabolic rate of oxygen (CMRO2 ) with MRI in the first 3 hours following the return of spontaneous circulation (ROSC) in a rat CA model. It was found that CBF, OEF and CMRO2 all revealed a time-dependent increase during the first 3 hours after the ROSC. Furthermore, the temporal trajectories of CBF and CMRO2 , but not OEF, were different across rats and related to neurologic outcomes at a later time (24 hours after the ROSC) (P < .001). Rats who manifested better outcomes revealed faster increases in CBF and CMRO2 during the acute stage. When investigating physiological parameters measured at a single time point, CBF (ρ = 0.82, P = .004) and CMRO2 (ρ = 0.80, P = .006) measured at ~ 3 hours post-ROSC were positively associated with neurologic outcome scores at 24 hours. These findings shed light on brain physiological changes following CA, and suggest that MRI measures of brain perfusion and metabolism may provide a potential biomarker to guide post-CA management.

Propylisopropylacetic acid (PIA), a constitutional isomer of valproic acid, uncompetitively inhibits arachidonic acid acylation by rat acyl-CoA synthetase 4: a potential drug for bipolar disorder.

BACKGROUND: Mood stabilizers used for treating bipolar disorder (BD) selectively downregulate arachidonic acid (AA) turnover (deacylation-reacylation) in brain phospholipids, when given chronically to rats. In vitro studies suggest that one of these, valproic acid (VPA), which is teratogenic, reduces AA turnover by inhibiting the brain long-chain acyl-CoA synthetase (Acsl)4 mediated acylation of AA to AA-CoA. We tested whether non-teratogenic VPA analogues might also inhibit Acsl4 catalyzed acylation, and thus have a potential anti-BD action. METHODS: Rat Acsl4-flag protein was expressed in Escherichia coli, and the ability of three VPA analogues, propylisopropylacetic acid (PIA), propylisopropylacetamide (PID) and N-methyl-2,2,3,3-tetramethylcyclopropanecarboxamide (MTMCD), and of sodium butyrate, to inhibit conversion of AA to AA-CoA by Acsl4 was quantified using Michaelis-Menten kinetics. RESULTS: Acsl4-mediated conversion of AA to AA-CoA in vitro was inhibited uncompetitively by PIA, with a Ki of 11.4mM compared to a published Ki of 25mM for VPA, while PID, MTMCD and sodium butyrate had no inhibitory effect. CONCLUSIONS: PIA's ability to inhibit conversion of AA to AA-CoA by Acsl4 in vitro suggests that, like VPA, PIA may reduce AA turnover in brain phospholipids in unanesthetized rats, and if so, may be effective as a non-teratogenic mood stabilizer in BD patients.

Valnoctamide, a non-teratogenic amide derivative of valproic acid, inhibits arachidonic acid activation in vitro by recombinant acyl-CoA synthetase-4.

OBJECTIVE: Valproic acid (VPA), a mood stabilizer used for treating bipolar disorder (BD), uncompetitively inhibits acylation of arachidonic acid (AA) by recombinant AA-selective acyl-CoA synthetase 4 (Acsl4) at an enzyme inhibition constant (Ki ) of 25 mM. Inhibition may account for VPA's ability to reduce AA turnover in brain phospholipids of unanesthetized rats and to be therapeutic in BD. However, VPA is teratogenic. We tested whether valnoctamide (VCD), a non-teratogenic amide derivative of a VPA chiral isomer, which had antimanic potency in a phase III BD trial, also inhibits recombinant Acsl4. METHODS: Rat Acsl4-flag protein was expressed in Escherichia coli. We used Michaelis-Menten kinetics to characterize and quantify the ability of VCD to inhibit conversion of AA to AA-CoA by recombinant Acsl4 in vitro. RESULTS: Acsl4-mediated activation of AA to AA-CoA by Acsl4 was inhibited uncompetitively by VCD, with a Ki of 6.38 mM. CONCLUSIONS: VCD's ability to uncompetitively inhibit AA activation to AA-CoA by Acsl4, at a lower Ki than VPA, suggests that, like VPA, VCD may reduce AA turnover in rat brain phospholipids. If so, VCD and other non-teratogenic Acsl4 inhibitors might be considered further for treating BD.

Mitochondria-Targeted Antioxidant Therapeutics for Traumatic Brain Injury.

Traumatic brain injury (TBI) is a major global health problem that affects both civilian and military populations worldwide. Post-injury acute, sub-acute, and chronic progression of secondary injury processes may contribute further to other neurodegenerative diseases. However, there are no approved therapeutic options available that can attenuate TBI-related progressive pathophysiology. Recent advances in preclinical research have identified that mitochondria-centric redox imbalance, bioenergetics failure and calcium dysregulation play a crucial role in secondary injury progression after TBI. Mitochondrial antioxidants play an important role in regulating redox homeostasis. Based on the proven efficacy of preclinical and clinical compounds and targeting numerous pathways to trigger innate antioxidant defense, we may be able to alleviate TBI pathology progression by primarily focusing on preserving post-injury mitochondrial and cerebral function. In this review, we will discuss novel mitochondria-targeted antioxidant compounds, which offer a high capability of successful clinical translation for TBI management in the near future.

Disturbed brain phospholipid and docosahexaenoic acid metabolism in calcium-independent phospholipase A(2)-VIA (iPLA(2)ß)-knockout mice.

Calcium-independent phospholipase A(2) group VIA (iPLA(2)ß) releases docosahexaenoic acid (DHA) from phospholipids in vitro. Mutations in the iPLA(2)ß gene, PLA2G6, are associated with dystonia-parkinsonism and infantile neuroaxonal dystrophy. To understand the role of iPLA(2)ß in brain, we applied our in vivo kinetic method using radiolabeled DHA in 4 to 5-month-old wild type (iPLA(2)ß(+/+)) and knockout (iPLA(2)ß(-/-)) mice, and measured brain DHA kinetics, lipid concentrations, and expression of PLA(2), cyclooxygenase (COX), and lipoxygenase (LOX) enzymes. Compared to iPLA(2)ß(+/+) mice, iPLA(2)ß(-/-) mice showed decreased rates of incorporation of unesterified DHA from plasma into brain phospholipids, reduced concentrations of several fatty acids (including DHA) esterified in ethanolamine- and serine-glycerophospholipids, and increased lysophospholipid fatty acid concentrations. DHA turnover in brain phospholipids did not differ between genotypes. In iPLA(2)ß(-/-) mice, brain levels of iPLA(2)ß mRNA, protein, and activity were decreased, as was the iPLA(2)γ (Group VIB PLA(2)) mRNA level, while levels of secretory sPLA(2)-V mRNA, protein, and activity and cytosolic cPLA(2)-IVA mRNA were increased. Levels of COX-1 protein were decreased in brain, while COX-2 protein and mRNA were increased. Levels of 5-, 12-, and 15-LOX proteins did not differ significantly between genotypes. Thus, a genetic iPLA(2)ß deficiency in mice is associated with reduced DHA metabolism, profound changes in lipid-metabolizing enzyme expression (demonstrating lack of redundancy) and of phospholipid fatty acid content of brain (particularly of DHA), which may be relevant to neurologic abnormalities in humans with PLA2G6 mutations.

Aging is associated with altered inflammatory, arachidonic acid cascade, and synaptic markers, influenced by epigenetic modifications, in the human frontal cortex.

Aging is a risk factor for Alzheimer's disease (AD) and is associated with cognitive decline. However, underlying molecular mechanisms of brain aging are not clear. Recent studies suggest epigenetic influences on gene expression in AD, as DNA methylation levels influence protein and mRNA expression in postmortem AD brain. We hypothesized that some of these changes occur with normal aging. To test this hypothesis, we measured markers of the arachidonic acid (AA) cascade, neuroinflammation, pro- and anti-apoptosis factors, and gene specific epigenetic modifications in postmortem frontal cortex from nine middle-aged [41 ± 1 (SEM) years] and 10 aged subjects (70 ± 3 years). The aged compared with middle-aged brain showed elevated levels of neuroinflammatory and AA cascade markers, altered pro and anti-apoptosis factors and loss of synaptophysin. Some of these changes correlated with promoter hypermethylation of brain derived neurotrophic factor (BDNF), cyclic AMP responsive element binding protein (CREB), and synaptophysin and hypomethylation of BCL-2 associated X protein (BAX). These molecular alterations in aging are different from or more subtle than changes associated with AD pathology. The degree to which they are related to changes in cognition or behavior during normal aging remains to be evaluated.

Chronic clozapine reduces rat brain arachidonic acid metabolism by reducing plasma arachidonic acid availability.

Chronic administration of mood stabilizers to rats down-regulates the brain arachidonic acid (AA) cascade. This down-regulation may explain their efficacy against bipolar disorder (BD), in which brain AA cascade markers are elevated. The atypical antipsychotics, olanzapine (OLZ) and clozapine (CLZ), also act against BD. When given to rats, both reduce brain cyclooxygenase activity and prostaglandin E(2) concentration; OLZ also reduces rat plasma unesterified and esterified AA concentrations, and AA incorporation and turnover in brain phospholipid. To test whether CLZ produces similar changes, we used our in vivo fatty acid method in rats given 10 mg/kg/day i.p. CLZ, or vehicle, for 30 days; or 1 day after CLZ washout. [1-(14) C]AA was infused intravenously for 5 min, arterial plasma was collected and high-energy microwaved brain was analyzed. CLZ increased incorporation coefficients ki * and decreased [corrected] rates J(in,i) of plasma unesterified AA into brain phospholipids. [corrected]. These effects disappeared after washout. Thus, CLZ and OLZ similarly down-regulated kinetics and cyclooxygenase expression of the brain AA cascade, likely by reducing plasma unesterified AA availability. Atypical antipsychotics and mood stabilizers may be therapeutic in BD by down-regulating, indirectly or directly respectively, the elevated brain AA cascade of that disease.

Quantification of Cerebral Vascular Autoregulation Immediately Following Resuscitation from Cardiac Arrest.

Cerebral vascular autoregulation is impaired following resuscitation from cardiac arrest (CA), and its quantification may allow assessing CA-induced brain injury. However, hyperemia occurring immediately post-resuscitation limits the application of most metrics that quantify autoregulation. Therefore, to characterize autoregulation during this critical period, we developed three novel metrics based on how the cerebrovascular resistance (CVR) covaries with changes in cerebral perfusion pressure (CPP): (i) Î¸CVR, which quantifies the CVR vs CPP gradient, (ii) a CVR-based transfer function analysis, and (iii) CVRx, the correlation coefficient between CPP and CVR. We tested these metrics in a model of asphyxia induced CA and resuscitation using seven adult male Wistar rats. Mean arterial pressure (MAP) and cortical blood flow recorded for 30 min post-resuscitation via arterial cannulation and laser speckle contrast imaging, were used as surrogates of CPP and cerebral blood flow (CBF), while CVR was computed as the CPP/CBF ratio. Using our metrics, we found that the status of cerebral vascular autoregulation altered substantially during hyperemia, with changes spread throughout the 0-0.05 Hz frequency band. Our metrics push the boundary of how soon autoregulation can be assessed, and if validated against outcome markers, may help develop a reliable metric of brain injury post-resuscitation.

Comprehensive evaluation of mitochondrial redox profile, calcium dynamics, membrane integrity and apoptosis markers in a preclinical model of severe penetrating traumatic brain injury.

Traumatic Brain Injury (TBI) is caused by the external physical assaults damages the brain. It is a heterogeneous disorder that remains a leading cause of death and disability in the military and civilian population of the United States. Preclinical investigations of mitochondrial responses in TBI have ascertained that mitochondrial dysfunction is an acute indicator of cellular damage and plays a pivotal role in long-term injury progression through cellular excitotoxicity. The current study was designed to provide an in-depth evaluation of mitochondrial endpoints with respect to redox and calcium homeostasis, and cell death responses following penetrating TBI (PTBI). To evaluate these pathological cascades, anesthetized adult male rats (N = 6/group) were subjected to either 10% unilateral PTBI or Sham craniectomy. Animals were euthanized at 24 h post-PTBI, and purified mitochondrial fractions were isolated from the brain injury core and perilesional areas. Overall, increased reactive oxygen and nitrogen species (ROS/RNS) production, and elevated oxidative stress markers such as 4-hydroxynonenal (4-HNE), 3-nitrotyrosine (3-NT), and protein carbonyls (PC) were observed in the PTBI group compared to Sham. Mitochondrial antioxidants such as glutathione, peroxiredoxin (PRX-3), thioredoxin (TRX), nicotinamide adenine dinucleotide phosphate (NADPH), superoxide dismutase (SOD), and catalase (CAT) levels were significantly decreased after PTBI. Likewise, PTBI mitochondria displayed significant loss of Ca2+ homeostasis, early opening of mitochondrial permeability transition pore (mPTP), and increased mitochondrial swelling. Both, outer and inner mitochondrial membrane integrity markers, such as voltage-dependent anion channels (VDAC) and cytochrome c (Cyt C) expression were significantly decreased following PTBI. The apoptotic cell death was evidenced by significantly decreased B-cell lymphoma-2 (Bcl-2) and increased glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression after PTBI. Collectively, current results highlight the comprehensive picture of mitochondria-centric acute pathophysiological responses following PTBI, which may be utilized as novel prognostic indicators of disease progression and theragnostic indicators for evaluating neuroprotection therapeutics following TBI.

Valproate uncompetitively inhibits arachidonic acid acylation by rat acyl-CoA synthetase 4: relevance to valproate's efficacy against bipolar disorder.

BACKGROUND: The ability of chronic valproate (VPA) to reduce arachidonic acid (AA) turnover in brain phospholipids of unanesthetized rats has been ascribed to its inhibition of acyl-CoA synthetase (Acsl)-mediated activation of AA to AA-CoA. Our aim was to identify a rat Acsl isoenzyme that could be inhibited by VPA in vitro. METHODS: Rat Acsl3-, Acsl6v1- and Acsl6v2-, and Acsl4-flag proteins were expressed in E. coli, and the ability of VPA to inhibit their activation of long-chain fatty acids to acyl-CoA was estimated using Michaelis-Menten kinetics. RESULTS: VPA uncompetitively inhibited Acsl4-mediated conversion of AA and of docosahexaenoic (DHA) but not of palmitic acid to acyl-CoA, but did not affect AA conversion by Acsl3, Acsl6v1 or Acsl6v2. Acsl4-mediated conversion of AA to AA-CoA showed substrate inhibition and had a 10-times higher catalytic efficiency than did conversion of DHA to DHA-CoA. Butyrate, octanoate, or lithium did not inhibit AA activation by Acsl4. CONCLUSIONS: VPA's ability to inhibit Acsl4 activation of AA and of DHA to their respective acyl-CoAs, when related to the higher catalytic efficiency of AA than DHA conversion, may account for VPA's selective reduction of AA turnover in rat brain phospholipids, and contribute to VPA's efficacy against bipolar disorder.

Systemic administration of dendrimer N-acetyl cysteine improves outcomes and survival following cardiac arrest.

Cardiac arrest (CA), the sudden cessation of effective cardiac pumping function, is still a major clinical problem with a high rate of early and long-term mortality. Post-cardiac arrest syndrome (PCAS) may be related to an early systemic inflammatory response leading to exaggerated and sustained neuroinflammation. Therefore, early intervention with targeted drug delivery to attenuate neuroinflammation may greatly improve therapeutic outcomes. Using a clinically relevant asphyxia CA model, we demonstrate that a single (i.p.) dose of dendrimer-N-acetylcysteine conjugate (D-NAC), can target "activated" microglial cells following CA, leading to an improvement in post-CA survival rate compared to saline (86% vs. 45%). D-NAC treatment also significantly improved gross neurological score within 4 h of treatment (p < 0.05) and continued to show improvement at 48 h (p < 0.05). Specifically, there was a substantial impairment in motor responses after CA, which was subsequently improved with D-NAC treatment (p < 0.05). D-NAC also mitigated hippocampal cell density loss seen post-CA in the CA1 and CA3 subregions (p < 0.001). These results demonstrate that early therapeutic intervention even with a single D-NAC bolus results in a robust sustainable improvement in long-term survival, short-term motor deficits, and neurological recovery. Our current work lays the groundwork for a clinically relevant therapeutic approach to treating post-CA syndrome.

Chronic olanzapine treatment decreases arachidonic acid turnover and prostaglandin E2 concentration in rat brain.

The atypical antipsychotic, olanzapine (OLZ), is used to treat bipolar disorder, but its therapeutic mechanism of action is not clear. Arachidonic acid (AA, 20:4n-6) plays a critical role in brain signaling and an up-regulated AA metabolic cascade was reported in postmortem brains from bipolar disorder patients. In this study, we tested whether, similar to the action of the mood stabilizers lithium, carbamazepine and valproate, chronic OLZ treatment would reduce AA turnover in rat brain. We administered OLZ (6 mg/kg/day) or vehicle i.p. to male rats once daily for 21 days. A washout group received 21 days of OLZ followed by vehicle on day 22. Two hours after the last injection, [1-¹4C]AA was infused intravenously for 5 min, and timed arterial blood samples were taken. After the rat was killed at 5 min, its brain was microwaved, removed and analyzed. Chronic OLZ decreased plasma unesterified AA concentration, AA incorporation rates and AA turnover in brain phospholipids. These effects were absent after washout. Consistent with reduced AA turnover, OLZ decreased brain cyclooxygenase activity and the brain concentration of the proinflammatory AA-derived metabolite, prostaglandin E2, In view of up-regulated brain AA metabolic markers in bipolar disorder, the abilities of OLZ and the mood stabilizers to commonly decrease prostaglandin E2, and AA turnover in rat brain phospholipids, albeit by different mechanisms, may be related to their efficacy against the disease.

Intranasal Orexin After Cardiac Arrest Leads to Increased Electroencephalographic Gamma Activity and Enhanced Neurologic Recovery in Rats.

OBJECTIVES: Prolonged cardiac arrest is known to cause global ischemic brain injury and functional impairment. Upon resuscitation, electroencephalographic recordings of brain activity begin to resume and can potentially be used to monitor neurologic recovery. We have previously shown that intrathecal orexin shows promise as a restorative drug and arousal agent in rodents. Our goal is to determine the electrophysiology effects of orexin in a rodent model of asphyxial cardiac arrest, focusing on the electroencephalographic activity in the gamma and super-gamma bands (indicative of return of higher brain function). DESIGN: Experimental animal study. SETTING: University-based animal research laboratory. SUBJECTS: Adult male Wistar rats. INTERVENTIONS: In an established model of asphyxial cardiac arrest (n = 24), we treated half of Wistar rats with orexin administered intranasally by atomizer 30 minutes post return of spontaneous circulation in one of two dose levels (10 and 50 µM); the rest were treated with saline as control. Continuous electroencephalographic recording was obtained and quantitatively analyzed for the gamma fraction. Gamma and high-frequency super-gamma band measures were compared against clinical recovery according to Neuro-Deficit Score. MEASUREMENTS AND MAIN RESULTS: Compared with the control cohort, the high-dose orexin cohort showed significantly better Neuro-Deficit Score 4 hours after return of spontaneous circulation (55.17 vs 47.58; p < 0.02) and significantly higher mean gamma fraction (0.251 vs 0.177; p < 0.02) in cerebral regions surveyed by rostral electrodes for the first 170 minutes after administration of orexin. CONCLUSIONS: Our findings support early and continuous monitoring of electroencephalography-based gamma activity as a marker of better functional recovery after intranasal administration of orexin as measured by Neuro-Deficit Score in an established animal model of asphyxial cardiac arrest.

Effect of treatment with cadmium on structure-function relationships in rat liver mitochondria: studies on oxidative energy metabolism and lipid/phospholipids profiles.

Effects of treatment with a single intraperitoneal injection of cadmium (Cd) on oxidative energy metabolism and lipid/phospholipid profiles of rat liver mitochondria were examined at the end of 1 week and 1 month. Following Cd treatment the body weight increased only in the 1 month group, whereas the liver weight increased in both groups. State 3 and 4 respiration rates in general decreased significantly, with the maximum effect being seen with succinate. The 1 week Cd group showed decreased respiratory activity with glutamate, pyruvate + malate, and succinate as the substrates. In the 1 month Cd-treated group respiration rates recovered with glutamate and pyruvate + malate but not with succinate. All cytochrome contents decreased in the 1 week Cd-treated group but recovered in the 1 month group. ATPase activity registered an increase in both Cd-treated groups. Dehydrogenase activities increased in the 1 week group but decreased in the 1 month Cd-treated group. The mitochondrial cholesterol content increased in the 1 week Cd-treated group. In the 1 week Cd-treated group the lysophospholipid (Lyso), sphingomyelin (SPM), and diphosphatidylglycerol (DPG) components increased. By contrast, the phosphatidylethanolamine (PE) component decreased. In the 1 month Cd-treated group the phosphatidylinositol, phosphatidylserine, and DPG components increased, whereas the Lyso, SPM, and phosphatidylcholine components decreased. The results demonstrate that single-dose Cd treatment can have adverse effects on liver mitochondrial oxidative energy metabolism and lipid/phosphopholipid profiles, which in turn can affect membrane structure-function relationships.

Therapeutic hypothermia promotes cerebral blood flow recovery and brain homeostasis after resuscitation from cardiac arrest in a rat model.

Laboratory and clinical studies have demonstrated that therapeutic hypothermia (TH), when applied as soon as possible after resuscitation from cardiac arrest (CA), results in better neurological outcome. This study tested the hypothesis that TH would promote cerebral blood flow (CBF) restoration and its maintenance after return of spontaneous circulation (ROSC) from CA. Twelve Wistar rats resuscitated from 7-min asphyxial CA were randomized into two groups: hypothermia group (7 H, n = 6), treated with mild TH (33-34℃) immediately after ROSC and normothermia group (7 N, n = 6,37.0 ± 0.5℃). Multiple parameters including mean arterial pressure, CBF, electroencephalogram (EEG) were recorded. The neurological outcomes were evaluated using electrophysiological (information quantity, IQ, of EEG) methods and a comprehensive behavior examination (neurological deficit score, NDS). TH consistently promoted better CBF restoration approaching the baseline levels in the 7 H group as compared with the 7 N group. CBF during the first 5-30 min post ROSC of the two groups was 7 H:90.5% ± 3.4% versus 7 N:76.7% ± 3.5% (P < 0.01). Subjects in the 7 H group showed significantly better IQ scores after ROSC and better NDS scores at 4 and 24 h. Early application of TH facilitates restoration of CBF back to baseline levels after CA, which in turn results in the restoration of brain electrical activity and improved neurological outcome.

Effect of treatment with cadmium on kinetic properties of Na(+), K(+)-ATPase and glucose-6-phosphatase activity in rat liver microsomes a correlative study on influence of lipid/phospholipid make-up.

Studies on Cd hepatotoxicity have focused mainly on induction of cytochrome P(450) system and related enzymes. In the present study young adult male rats given a single intra-peritoneal injection of Cd (0.84mg Cd/kg body weight) and effects on kinetic parameters rat liver microsomal Na(+), K(+)-ATPase and G6Pase were evaluated at the end of 1 month and 1 week. The substrate and temperature kinetics parameters were examined and attempts were made to seek correlation with changes in lipid/phospholipid profiles. The Na(+), K(+) ATPase activity decreased only in 1 week Cd-treated group but recovered at the end of 1 month. The activity resolved in two distinct kinetic components in control as well as the experimental groups. In 1 week Cd-treated group the K(m) value of both the components was unchanged, whereas V(max) value decreased. In 1-month Cd-treated group V(max) value only of component I increased. The catalytic efficiency of both the components was not affected in the experimental groups. In 1-week Cd-treated group the energy of activations at high-temperature range (E(H)) and low-temperature range (E(L)) decreased, whereas for 1-month Cd-treated group the energies of activations did not change. The G6Pase activity measured at 37 degrees C was high only in 1-month Cd-treated group. The activity resolved in two kinetically distinguishable components in control as well as in the experimental groups. K(m) value of component I decreased in both the Cd-treated groups. In 1-month Cd-treated group the V(max) value of component II increased. The catalytic efficiency of G6Pase was unchanged despite changes in K(m) and V(max). In 1-week Cd-treated group the E(H) and E(L) decreased, whereas only E(L) showed decrease in 1-month Cd-treated group. Cholesterol (CHL) content increased in both the Cd-treated groups. Content of lysophospholipid (Lyso), spinghomyelin (SPM) and phosphatidic acid (PA) increased, whereas phosphatidylcholine (PC) and phosphatidylserine (PS) decreased in 1-week Cd-treated group. In 1-month Cd group the Lyso, SPM, and PC increased while PC, phosphatidylethanolamine (PE) and PA decreased. In conclusion, Cd has short-term effects on microsomal Na(+), K(+)-ATPase which are reversed by the end of 1 month and that G6Pase does not seem to be a target of Cd insult.

Thyroidal regulation of substrate kinetics properties of cytochrome oxidase in rat liver mitochondria.

Effects of thyroidectomy (T(x)) and subsequent treatment with 3,5,3'-triiodothyronine (T(3)), and combined treatment (T(R)) with T(3) + thyroxine (T(4)) on substrate kinetics properties of cytochrome oxidase of rat liver mitochondria were examined. T(x) resulted in lowering of cytochromes content with decrease in the enzyme activity, and K(m) and V(max). T(3) and T(R) regimens restored the cytochromes contents and the V(max) values to normal. In control, T(3) and T(R) groups the enzyme activity resolved in two kinetic components; in T(x) group three kinetic components were evident. The K(m) values for all components decreased significantly in the experimental groups with concomitant increase in catalytic efficiency, K(cat)/K(m). Significant alterations in the contents of total phospholipid and of cholesterol were noted while the changes in the phospholipids composition were only of restricted nature. Regression analysis revealed that total phospholipid, cholesterol and phosphatidylcholine, phosphatidylethanolamine play significant role in fine tuning the enzyme activity.