Cerebrospinal fluid (CSF) proteins may be useful biomarkers of neuronal death and ultimate prognosis after hypoxic-ischemic brain injury. Cytochrome c has been identified in the CSF of children following traumatic brain injury. Cytochrome c is required for cellular respiration but it is also a central component of the intrinsic pathway of apoptosis. Thus, in addition to serving as a biomarker, cytochrome c release into CSF may have an effect upon survival of adjacent neurons. In this study, we use Western blot and ELISA to show that cytochrome c is elevated in CSF obtained from pediatric rats following resuscitation from cardiac arrest. Using biotinylated human cytochrome c in culture media we show that cytochrome c crosses the cell membrane and is incorporated into mitochondria of neurons exposed to anoxia. Lastly, we show that addition of human cytochrome c to primary neuronal culture exposed to anoxia improves survival. To our knowledge, this is the first study to show cytochrome c is elevated in CSF following hypoxic ischemic brain injury. Results from primary neuronal culture suggest that extracellular cytochrome c is able to cross the cell membrane of injured neurons, incorporate into mitochondria, and promote survival following anoxia.
cerebrospinal fluid; brain ischemia; cardiac arrest; cytochrome c
Hypotension after traumatic brain injury (TBI) worsens outcome. We published the first report of TBI plus hemorrhagic shock (HS) in mice using a volume-controlled approach and noted increased neuronal death. To rigorously control blood pressure during HS, a pressure-controlled HS model is required. Our hypothesis was that a brief, severe period of pressure-controlled HS after TBI in mice will exacerbate functional deficits and neuropathology versus TBI or HS alone. C57BL6 male mice were randomized into four groups (n=10/group): sham, HS, controlled cortical impact (CCI), and CCI+HS. We used a pressure-controlled shock phase (mean arterial pressure [MAP]=25–27 mm Hg for 35 min) and its treatment after mild to moderate CCI including, a 90 min pre-hospital phase, during which lactated Ringer's solution was given to maintain MAP >70 mm Hg, and a hospital phase, when the shed blood was re-infused. On days 14–20, the mice were evaluated in the Morris water maze (MWM, hidden platform paradigm). On day 21, the lesion and hemispheric volumes were quantified. Neuropathology and hippocampal neuron counts (hematoxylin and eosin [H&E], Fluoro-Jade B, and NeuN) were evaluated in the mice (n=60) at 24 h, 7 days, or 21 days (n=5/group/time point). HS reduced MAP during the shock phase in the HS and CCI+HS groups (p<0.05). Fluid requirements during the pre-hospital phase were greatest in the CCI+HS group (p<0.05), and were increased in HS versus sham and CCI animals (p<0.05). MWM latency was increased on days 14 and 15 after CCI+HS (p<0.05). Swim speed and visible platform latency were impaired in the CCI+HS group (p<0.05). CCI+HS animals had increased contusion volume versus the CCI group (p<0.05). Hemispheric volume loss was increased 33.3% in the CCI+HS versus CCI group (p<0.05). CA1 cell loss was seen in CCI+HS and CCI animals at 24 h and 7 days (p<0.05). CA3 cell loss was seen after CCI+HS (p<0.05 at 24 h and 7 days). CA1 cell loss at 21 days was seen only in CCI+HS animals (p<0.05). Brief, severe, pressure-controlled HS after CCI produces robust functional deficits and exacerbates neuropathology versus CCI or HS alone.
blast injury; controlled cortical impact; head injury; head trauma; Morris water maze; polytrauma; secondary insult
High-mobility group box 1 (HMGB1) is a ubiquitous nuclear protein that is passively released from damaged and necrotic cells, and actively released from immune cells. In contrast, cytochrome c is released from mitochondria in apoptotic cells, and is considered a reliable biomarker of apoptosis. Thus, HMGB1 and cytochrome c may in part reflect the degree of necrosis and apoptosis present after traumatic brain injury (TBI), where both are felt to contribute to cell death and neurological morbidity. Ventricular cerebrospinal fluid (CSF) was obtained from children admitted to the intensive care unit (ICU) after TBI (n=37). CSF levels of HMGB1 and cytochrome c were determined at four time intervals (0–24 h, 25–48 h, 49–72 h, and>72 h after injury) using enzyme-linked immunosorbent assay (ELISA). Lumbar CSF from children without TBI served as controls (n=12). CSF HMGB1 levels were: control=1.78±0.29, 0–24 h=5.73±1.45, 25–48 h=5.16±1.73, 49–72 h=4.13±0.75,>72 h=3.80±0.90 ng/mL (mean±SEM). Peak HMGB1 levels were inversely and independently associated with favorable Glasgow Outcome Scale (GOS) scores at 6 mo (0.49 [0.24–0.97]; OR [5–95% CI]). CSF cytochrome c levels were: control=0.37±0.10, 0–24 h=0.69±0.15, 25–48 h=0.82±0.48, 49–72 h=1.52±1.08,>72 h=1.38±1.02 ng/mL (mean±SEM). Peak cytochrome c levels were independently associated with abusive head trauma (AHT; 24.29 [1.77–334.03]) and inversely and independently associated with favorable GOS scores (0.42 [0.18–0.99]). In conclusion, increased CSF levels of HMGB1 and cytochrome c were associated with poor outcome after TBI in infants and children. These data are also consistent with the designation of HMGB1 as a “danger signal.” Distinctly increased CSF cytochrome c levels in infants and children with AHT and poor outcome suggests that apoptosis may play an important role in this unique patient population.
abusive head trauma; child abuse; cytochrome c; high mobility group box 1
The relationship between the timing of the initiation of antiretroviral therapy (ART) after infection with human immunodeficiency virus type 1 (HIV-1) and the recovery of CD4+ T-cell counts is unknown.
In a prospective, observational cohort of persons with acute or early HIV-1 infection, we determined the trajectory of CD4+ counts over a 48-month period in partially overlapping study sets: study set 1 included 384 participants during the time window in which they were not receiving ART and study set 2 included 213 participants who received ART soon after study entry or sometime thereafter and had a suppressed plasma HIV viral load. We investigated the likelihood and rate of CD4+ T-cell recovery to 900 or more cells per cubic millimeter within 48 months while the participants were receiving viral-load–suppressive ART.
Among the participants who were not receiving ART, CD4+ counts increased spontaneously, soon after HIV-1 infection, from the level at study entry (median, 495 cells per cubic millimeter; interquartile range, 383 to 622), reached a peak value (median, 763 cells per cubic millimeter; interquartile range, 573 to 987) within approximately 4 months after the estimated date of infection, and declined progressively thereafter. Recovery of CD4+ counts to 900 or more cells per cubic millimeter was seen in approximately 64% of the participants who initiated ART earlier (≤4 months after the estimated date of HIV infection) as compared with approximately 34% of participants who initiated ART later (>4 months) (P<0.001). After adjustment for whether ART was initiated when the CD4+ count was 500 or more cells per cubic millimeter or less than 500 cells per cubic millimeter, the likelihood that the count would increase to 900 or more cells per cubic millimeter was lower by 65% (odds ratio, 0.35), and the rate of recovery was slower by 56% (rate ratio, 0.44), if ART was initiated later rather than earlier. There was no association between the plasma HIV RNA level at the time of initiation of ART and CD4+ T-cell recovery.
A transient, spontaneous restoration of CD4+ T-cell counts occurs in the 4-month time window after HIV-1 infection. Initiation of ART during this period is associated with an enhanced likelihood of recovery of CD4+ counts. (Funded by the National Institute of Allergy and Infectious Diseases and others.)
Caffeine, the most widely consumed psychoactive drug and a weak adenosine receptor antagonist, can be neuroprotective or neurotoxic depending on the experimental model or neurologic disorder. However, its contribution to pathophysiology and outcome in traumatic brain injury (TBI) in humans is undefined. We assessed serial cerebrospinal fluid (CSF) concentrations of caffeine and its metabolites (theobromine, paraxanthine, and theophylline) by high-pressure liquid chromatography/ultraviolet in 97 ventricular CSF samples from an established bank, from 30 adults with severe TBI. We prospectively selected a threshold caffeine level of ≥1 μmol/L (194 ng/mL) as clinically significant. Demographics, Glasgow Coma Scale (GCS) score, admission blood alcohol level, and 6-month dichotomized Glasgow Outcome Scale (GOS) score were assessed. Mean time from injury to initial CSF sampling was 10.77±3.13 h. On initial sampling, caffeine was detected in 24 of 30 patients, and the threshold was achieved in 9 patients. Favorable GOS was seen more often in patients with CSF caffeine concentration ≥ versus < the threshold (55.6 versus 11.8%, P = 0.028). Gender, age, admission CGS score, admission blood alcohol level, and admission systolic arterial blood pressure did not differ between patients with CSF caffeine concentration ≥ versus < the threshold. Increases in CSF concentrations of the caffeine metabolites theobromine and paraxanthine were also associated with favorable outcome (P = 0.018 and 0.056, respectively). Caffeine and its metabolites are commonly detected in CSF in patients with severe TBI and in an exploratory assessment are associated with favorable outcome. We speculate that caffeine may be neuroprotective by long-term upregulation of adenosine A1 receptors or acute inhibition of A2a receptors.
adenosine; alcohol; coffee; head injury; head trauma; theobromine
Chronic elevation of angiotensin (Ang)-II can lead to myocardial inflammation, hypertrophy and cardiac failure. The adaptor molecule CIKS (connection to IKK and SAPK/JNK) activates the IκB kinase/nuclear factor (NF)-κB and JNK/activator protein (AP)-1 pathways in autoimmune and inflammatory diseases. Since Ang-II is a potent activator of NF-κB and AP-1, we investigated whether CIKS is critical in Ang-II-mediated cardiac hypertrophy. Here we report that Ang-II induced CIKS mRNA and protein expression, CIKS binding to IKK and JNK perhaps functioning as a scaffold protein, CIKS-dependent IKK/NF-κB and JNK/AP-1 activation, p65 and c-Jun phosphorylation and nuclear translocation, NF-κB- and AP-1-dependent IL-18 and MMP-9 induction, and hypertrophy of adult cardiomyocytes isolated from WT, but not CIKS-null mice. These results were recapitulated in WT-cardiomyocytes following CIKS knockdown. Infusion of Ang-II for 7 days induced cardiac hypertrophy, increased collagen content, and upregulated CIKS mRNA and protein expression in WT mice, whereas cardiac hypertrophy and collagen deposition were markedly attenuated in the CIKS-null mice, despite a similar increase in systolic blood pressure and DPI-inhibitable superoxide generation in both types of animals. Further, Ang-II-induced IKK/p65 and JNK/c-Jun phosphorylation, NF-κB and AP-1 activation, and IL-18 and MMP-9 expression were also markedly attenuated in CIKS-null mice. These results demonstrate that CIKS is critical in Ang-II-induced cardiomyocyte hypertrophy and fibrosis, and that CIKS is an important intermediate in Ang-II induced redox signaling. CIKS is a potential therapeutic target in cardiac hypertrophy, fibrosis, and congestive heart failure.
RAAS; NADPH oxidase; Act1; TRAF3IP2; fibrosis; cardiac hypertrophy
Brain contains a highly diversified complement of molecular species of a mitochondria-specific phospholipid, cardiolipin (CL), which - due to its polyunsaturation - can readily undergo oxygenation. Here, we used global lipidomics analysis in experimental traumatic brain injury (TBI) and showed that TBI was accompanied by oxidative consumption of polyunsaturated CL and accumulation of more than 150 new oxygenated molecular species in CL. RNAi-based manipulations of CL-synthase and CL levels conferred resistance of primary rat cortical neurons to mechanical stretch - an in vitro model of traumatic neuronal injury. By applying the novel brain permeable mitochondria-targeted electron-scavenger, we prevented CL oxygenation in the brain, achieved a substantial reduction in neuronal death both in vitro and in vivo, and markedly reduced behavioral deficits and cortical lesion volume. We conclude that CL oxygenation generates neuronal death signals and that its prevention by mitochondria-targeted small molecule inhibitors represents a new target for neuro-drug discovery.
Drowning is a leading cause of accidental death. Survivors may sustain severe neurologic morbidity. There is negligible research specific to brain injury in drowning making current clinical management non-specific to this disorder. This review represents an evidence-based consensus effort to provide recommendations for management and investigation of the drowning victim. Epidemiology, brain-oriented prehospital and intensive care, therapeutic hypothermia, neuroimaging/monitoring, biomarkers, and neuroresuscitative pharmacology are addressed. When cardiac arrest is present, chest compressions with rescue breathing are recommended due to the asphyxial insult. In the comatose patient with restoration of spontaneous circulation, hypoxemia and hyperoxemia should be avoided, hyperthermia treated, and induced hypothermia (32–34 °C) considered. Arterial hypotension/hypertension should be recognized and treated. Prevent hypoglycemia and treat hyperglycemia. Treat clinical seizures and consider treating non-convulsive status epilepticus. Serial neurologic examinations should be provided. Brain imaging and serial biomarker measurement may aid prognostication. Continuous electroencephalography and N20 somatosensory evoked potential monitoring may be considered. Serial biomarker measurement (e.g., neuron specific enolase) may aid prognostication. There is insufficient evidence to recommend use of any specific brain-oriented neuroresuscitative pharmacologic therapy other than that required to restore and maintain normal physiology. Following initial stabilization, victims should be transferred to centers with expertise in age-specific post-resuscitation neurocritical care. Care should be documented, reviewed, and quality improvement assessment performed. Preclinical research should focus on models of asphyxial cardiac arrest. Clinical research should focus on improved cardiopulmonary resuscitation, re-oxygenation/reperfusion strategies, therapeutic hypothermia, neuroprotection, neurorehabilitation, and consideration of drowning in advances made in treatment of other central nervous system disorders.
Drowning; Brain; Asphyxia; Cardiac arrest
To determine the relationship between hyperglycemia and outcome in infants and children after severe traumatic brain injury (TBI)
Retrospective review of a prospectively-collected Pediatric Neurotrauma Registry
Setting and Patients
Children admitted after severe TBI (post-resuscitation GCS ≤ 8) were studied (1999 – 2004). A subset of children (n = 28) were concurrently enrolled in a randomized, controlled clinical trial of early hypothermia for neuroprotection
Demographic data, serum glucose concentrations and outcome assessments were collected
Methods and Main Results
Children (n = 57) were treated with a standard TBI protocol. Exogenous glucose was withheld for 48 h after injury unless hypoglycemia was observed (blood glucose < 70 mg/dl). Early (first 48 h) and Late (49 - 168 h) time periods were defined and mean blood glucose concentrations were calculated. Additionally, children were categorized based on peak blood glucose concentrations during each time period (normal (NG) – blood glucose < 150 mg/dl; mild hyperglycemia (MHG) – blood glucose ≤ 200 mg/dl; severe hyperglycemia (SHG) – blood glucose > 200 mg/dl). In the Late period, an association between elevated mean serum glucose concentration and outcome was observed (133.5 ± 5.6 mg/dl in the unfavorable group vs. 115.4 ± 4.1 mg/dl in favorable group, p = 0.02). This association continued to be significant after correcting for injury severity, age, and exposure to insulin (p = 0.03). Similarly, in the Late period, children within the SHG had decreased incidence of good outcome compared to children within the other glycemic groups (% good outcome: NG – 61.9%, MHG – 73.7%, SHG – 33.3%; p = 0.05). However, when adjusted for exposure to insulin, this relationship was no longer statistically significant.
In children with severe TBI, hyperglycemia beyond the initial 48 hours is associated with poor outcome. This relationship was observed in both our analysis of mean blood glucose concentrations as well as amongst the patients with episodic severe hyperglycemia. This observation suggests a relationship between hyperglycemia and outcome from TBI. However, only a prospective study can answer the important question, whether manipulating serum glucose concentration can improve outcome after TBI in children
To compare the correlation of intracranial pressure (ICP) measurement and time to detection of ICP crises (defined as ICP ≥ 20 mm Hg for ≥ 5 min) between an intraparenchymal (IP) monitor and external ventricular drain (EVD) in children where continuous cerebrospinal fluid (CSF) diversion was used as a therapy for severe traumatic brain injury (TBI).
Academic, pediatric intensive care unit.
Retrospective review of a prospectively-collected Pediatric Neurotrauma database.
Children with severe TBI (GCS ≤ 8) who underwent ICP monitoring with both IP and EVD techniques were studied. In Cohort 1 (n = 58), hourly ICP measurements were extracted from the medical record. In Cohort 2 (n = 4), ICP measurements were collected every minute by an automated data collection system.
Measurements and Main Results
The mean absolute difference in ICP (|ICP|) and intraclass correlation coefficients (ICC) were calculated. Timing to detection of ICP crises was analyzed. Data expressed as mean ± SEM. In cohort 1, 7,387 hours of data were analyzed and 399 hours (23,940 min) were analyzed in Cohort 2. In Cohort 1, |ICP| = 3.10 ± 0.04 mm Hg (ICC = 0.98, p < 0.001). |ICP| in Cohort 2 was 3.30 ± 0.05 mm Hg (ICC = 0.98, p < 0.001). In Cohort 2, a total of 75 ICP crises were observed. Fifty-five (73%) were detected first by the IP monitor, of which 35 were not identified by the EVD monitor. Time between IP and EVD detection of a crisis was 12.60 ± 2.34 min.
EVD and IP measurements of ICP were highly correlated, although intermittent EVD ICP measurements may fail to identify ICP events when continuously draining CSF. In institutions using continuous CSF diversion as a therapy, a two-monitor system may be valuable for accomplishing monitoring and therapeutic goals.
Experimental evidence suggests that oxidative and nitrative mechanisms account for much of the dopaminergic neuronal injury in Parkinson’s disease (PD). The ubiquitously expressed non-receptor tyrosine kinase c-Abl is activated by oxidative stress and thus, may play a role in redox-mediated neurodegeneration. Recently, we reported that c-Abl is activated in PD and that a c-Abl inhibitor mitigated neuronal damage in a PD animal model, suggesting a novel neuroprotective therapeutic approach. In the studies presented here, we evaluated the efficacy of a potent and clinically relevant second-generation irreversible Abl kinase inhibitor, INNO-406, as a therapeutic agent for PD. Our studies reveal that INNO-406 is capable of preventing the progression of dopaminergic neuronal damage in a toxin-induced C57 mouse model of PD. Using bovine brain microvessel endothelium as an in vitro blood-brain barrier (BBB) model, we detected rapid and significant transfer of INNO-406. Additionally, pharmacokinetic analyses demonstrated significant nanomolar concentrations of INNO-406 in brain in the presence or absence of MPTP administration, however, INNO-406 did not alter the brain levels of MPP+ in MPTP-treated mice. Finally, we showed that 10 mg/kg of INNO-406 given to C57 mice for one week before MPTP treatment (4×20 mg/kg i.p., every 2 h) and then for one week after MPTP treatment decreased the loss of dopamine in the striatum by 45% and the loss of TH+ neurons in substantia nigra pars compacts by 40%. This treatment regimen also abrogated activation of c-Abl, tyrosine phosphorylation of the Abl substrate and E3-ubiquitin ligase parkin, and accumulation of the toxic parkin substrate AIMP2. We propose that compounds of the INNO-406 class of Abl inhibitors will be useful new neuroprotective drugs for the treatment of PD-like pathology in preclinical systems that should be easily translated to the clinic.
To describe the use and feasibility of therapeutic hypothermia after pediatric cardiac arrest.
Retrospective cohort study.
Pediatric tertiary care university hospital.
Infants and children (age 1 wk to 21 yrs) without complex congenital heart disease with return of spontaneous circulation after in-hospital or out-of-hospital cardiac arrest from 2000 to 2006.
Measurements and Main Results
We studied 181 patients after cardiac arrest, of which 91% were asphyxial in etiology (vs. cardiac) and 52% occurred in-hospital. Overall survival to hospital discharge was 45%. Forty patients received therapeutic hypothermia; all were admitted during or after 2002. Sixty percent of patients in the therapeutic hypothermia group had an initial temperature <35°C. The median therapeutic hypothermia target temperature was 34.0°C (33.5–34.8°C), was reached by 7 hrs (5–8 hrs) after admission in patients who were not hypothermic on admission, and was maintained for 24 hrs (16–48 hrs). Re-warming lasted 6 hrs (5–8 hrs). In the therapeutic hypothermia group, temperature <32°C occurred in 15% of patients and was associated with higher hospital mortality (29% vs. 11%; p = .02). Patients treated with therapeutic hypothermia differed from those treated with standard therapy, with more un-witnessed cardiac arrest (p = .04), more doses of epinephrine to achieve return of spontaneous circulation (p = .03), and a trend toward more out-of-hospital cardiac arrests (p = .11). After arrest, therapeutic hypothermia patients received more frequent electrolyte supplementation (p < .05). Standard therapy patients were twice as likely as therapeutic hypothermia patients to have a fever (>38°C) after arrest (37% vs. 18%; p = .02) and trended toward a higher rate of re-arrest (26% vs. 13%; p = .09). Rates of red blood cell transfusions, infection, and arrhythmias were similar between groups. There was no difference in hospital mortality (55.0% therapeutic hypothermia vs. 55.3% standard therapy; p = 1.0), and 78% of the therapeutic hypothermia survivors were discharged home (vs. 68% of the standard therapy survivors; p = .46). In multivariate analysis, mortality was independently associated with initial hypoglycemia or hyperglycemia, number of doses of epinephrine during resuscitation, asphyxial etiology, and longer duration of cardiopulmonary resuscitation, but not treatment group (odds ratio for mortality in the therapeutic hypothermia group, 0.47; p = .2).
This is the largest study reported on the use of therapeutic mild hypothermia in pediatric cardiac arrest to date. We found that therapeutic hypothermia was feasible, with target temperature achieved in <3 hrs overall. Temperature below target range was associated with increased mortality. Prospective study is urgently needed to determine the efficacy of therapeutic hypothermia in pediatric patients after cardiac arrest.
brain injury; cardiopulmonary resuscitation; cardiac arrest; pediatric; outcome
To fully describe gene expression dynamics requires the ability to quantitatively capture expression in individual cells over time. Automated systems for acquiring and analyzing real-time images are needed to obtain unbiased data across many samples and conditions. We developed a microfluidics device, the RootArray, in which 64 Arabidopsis thaliana seedlings can be grown and their roots imaged by confocal microscopy over several days without manual intervention. To achieve high throughput, we decoupled acquisition from analysis. In the acquisition phase, we obtain images at low magnification and segment to identify regions of interest. Coordinates are communicated to the microscope to record the regions of interest at high magnification. In the analysis phase, we reconstruct 3D objects from stitched high magnification images, and extract quantitative measurements from a virtual medial section of the root. We tracked hundreds of roots to capture detailed expression patterns of 12 transgenic reporter lines under different conditions.
We explored multiple quantitative measures of horizontal rectus extraocular muscle (EOM) morphology to determine the magnetic resonance imaging (MRI) measure best correlating with duction and thus contractility.
Surface coil coronal MRI was obtained in target-controlled central gaze and multiple positions of adduction and abduction in 26 orbits of 15 normal volunteers. Duction angles were determined by position changes of the globe-optic nerve junction. Cross-sectional areas, partial volumes, and location of peak cross-sections of the horizontal rectus EOMs were computed in contiguous image planes 2-mm thick spanning the EOM origins to the globe equator.
All measures correlated significantly with duction angle (P < 0.0001). The best measures obtainable in single image planes were the maximum change in the cross-sectional area between equivalent image planes, with coefficients of determination R2 = 0.92 for medial rectus (MR) and 0.91 for lateral rectus (LR), and percentage change in maximum cross-section with R2 = 0.79 for MR and 0.78 for LR. The best partial volume measure of contractility was the change in partial volumes in four contiguous posterior planes (R2 = 0.86 MR and for 0.89 LR), particularly when combined with the corresponding change in partial volume for the antagonist EOM (R2 = 0.95 for MR and LR).
EOM morphologic changes are highly correlated with degrees of duction and thus contractility. Both changes in single-plane maximum cross-sectional areas and posterior partial volumes provide accurate, quantitative measures of EOM contractility.
Magnetic resonance morphometry of horizontal rectus extraocular muscles is highly correlated with duction angle in normal subjects, suggesting that local volume and cross-section muscle features reflect contractile state.
Mechanical injury of neurites accompanied by rupture of mitochondrial membranes may lead to immediate nonspecific release and spreading of pro-apoptotic factors and activation of proteases, that is, execution of apoptotic program. In the current work, we studied the time course of the major biomarkers of apoptosis as they are induced by exposure of rat cortical neurons to mechanical stretch. By using transmission electron microscopy, we found that mitochondria in the neurites were damaged early (1 h) after mechanical stretch injury whereas somal mitochondria were significantly more resistant and demonstrated structural damage and degenerative mitochondrial changes at a later time point after stretch (12 h). We also report that the stretch injury caused immediate activation of reactive oxygen species production followed by selective oxidation of a mitochondria-specific phospholipid, cardiolipin, whose individual peroxidized molecular species have been identified and quantified by electrospray ionization mass spectrometry analysis. Most abundant neuronal phospholipids – phosphatidylcholine, phophatidylethanolamine – did not undergo oxidative modification. Simultaneously, a small-scale release of cytochrome c was observed. Notably, caspase activation and phosphatidylserine externalization – two irreversible apoptotic events designating a point of no return – are substantially delayed and do not occur until 6–12 h after the initial impact. The early onset of reactive oxygen species production and cytochrome c release may be relevant to direct stretch-induced damage to mitochondria. The delayed emergence of apoptotic neuronal death after the immediate mechanical damage to mitochondria suggests a possible window of opportunity for targeted therapies.
cardiolipin; in vitro traumatic brain injury; mitochondria; oxidative lipidomics; oxidative stress
Postresuscitation cerebral blood flow (CBF) disturbances and generation of reactive oxygen species likely contribute to impaired neurologic outcome after pediatric cardiac arrest (CA). Hence, we determined the effects of the antioxidant colloid polynitroxyl albumin (PNA) versus albumin or normal saline (NS) on CBF and neurologic outcome after asphyxial CA in immature rats. We induced asphyxia for 9 minutes in male and female postnatal day 16 to 18 rats randomized to receive PNA, albumin, or NS at resuscitation from CA or sham surgery. Regional CBF was measured serially from 5 to 150 minutes after resuscitation by arterial spin-labeled magnetic resonance imaging. We assessed motor function (beam balance and inclined plane), spatial memory retention (water maze), and hippocampal neuronal survival. Polynitroxyl albumin reduced early hyperemia seen 5 minutes after CA. In contrast, albumin markedly increased and prolonged hyperemia. In the delayed period after resuscitation (90 to 150 minutes), CBF was comparable among groups. Both PNA- and albumin-treated rats performed better in the water maze versus NS after CA. This benefit was observed only in males. Hippocampal neuron survival was similar between injury groups. Treatment of immature rats with PNA or albumin resulted in divergent acute changes in CBF, but both improved spatial memory retention in males after asphyxial CA.
anoxia; CBF autoregulation; cardiac arrest; global ischemia; oxidative stress
The pigmented Long-Evans rat has proven to be an excellent subject for studying visually guided behavior including quantitative visual psychophysics. This observation, together with its experimental accessibility and its close homology to the mouse, has made it an attractive model system in which to dissect the thalamic and cortical circuits underlying visual perception. Given that visually guided behavior in the absence of primary visual cortex has been described in the literature, however, it is an empirical question whether specific visual behaviors will depend on primary visual cortex in the rat. Here we tested the effects of cortical lesions on performance of two-alternative forced-choice visual discriminations by Long-Evans rats. We present data from one highly informative subject that learned several visual tasks and then received a bilateral lesion ablating >90% of primary visual cortex. After the lesion, this subject had a profound and persistent deficit in complex image discrimination, orientation discrimination, and full-field optic flow motion discrimination, compared with both pre-lesion performance and sham-lesion controls. Performance was intact, however, on another visual two-alternative forced-choice task that required approaching a salient visual target. A second highly informative subject learned several visual tasks prior to receiving a lesion ablating >90% of medial extrastriate cortex. This subject showed no impairment on any of the four task categories. Taken together, our data provide evidence that these image, orientation, and motion discrimination tasks require primary visual cortex in the Long-Evans rat, whereas approaching a salient visual target does not.
Metastatic dissemination in prostate cancer is often early, however not all cancer cells form clinical metastases. Map kinase kinase 4 (MKK4) suppresses metastasis in a preclinical prostate cancer model. We hypothesize that MKK4 will specifically inhibit metastatic colonization through impaired proliferation.
Three highly metastatic rat prostate cancer cell lines (AT6.1, Mat-Lu, AT3.1) were employed. Stably over-expressing HA-MKK4 or vector control lines were injected into immunocomprimised mice. These experiments validated that HA-MKK4 specifically affects metastatic colonization and increases survival. Median survival (days) with HA-MKK4 vs. vector was 42 vs. 28 (p<0.0001) for AT6.1, 25 vs. 19 (p<0.0001) for Mat-Lu and 27 vs. 20 (p<0.0001) for AT3.1. HA-MKK4 suppresses colonization within 14 days post dissemination, after which, exponential proliferation resumes. Although overt metastases retain HA-MKK4, it is inactive within these lesions. Nonetheless, metastasis derived cell lines were shown to retain functional HA-MKK4 and like their parental HA-MKK4 line, are suppressed for experimental metastasis formation in vivo. Disseminated AT6.1-HA-MKK4 cells were analyzed and were found to have an alteration in cell cycle. Specifically, there was an accumulation of cells in G1-phase (p=0.024), and decrease in S-phase (p=0.037) compared to vector.
In multiple prostate cancer lines, HA-MKK4 suppresses an early step in metastatic colonization. These data support a model in which MKK4 activation at the metastatic site causes a cell-cycle arrest, which is eventually overcome despite presence of functional HA-MKK4. Further studies will specifically interrogate the regulation of MKK4 activation within the metastatic microenvironment and the down-stream molecular events critical for metastasis suppression.
metastasis suppressor; metastatic colonization; prostate cancer; cell cycle
The resting membrane potential, Em, of mammalian cells is a fundamental physiological parameter. Even small changes in Em can modulate excitability, contractility and rates of cell migration. At present accurate, reproducible measurements of Em and determination of its ionic basis remain significant challenges when patch clamp methods are applied to small cells. In this study, a mathematical model has been developed which incorporates many of the main biophysical principles which govern recordings of the resting potential of “small cells”. Such a prototypical cell (approx. capacitance, 6 pF; input resistance 5 GΩ) is representative of neonatal cardiac myocytes, and other cells in the cardiovascular system (endothelium, fibroblasts) and small cells in other tissues, e.g., bone (osteoclasts) articular joints (chondrocytes) and the pancreas (β cells). Two common experimental conditions have been examined: (1) when the background K+ conductance is linear; and (2) when this K+ conductance is highly nonlinear and shows pronounced inward rectification. In the case of a linear K+ conductance, the presence of a “leakage” current through the seal resistance between the cell membrane and the patch pipette always depolarizes Em. Our calculations confirm that accurate characterization of Em is possible when the seal resistance is at least five times larger than the input resistance of the targeted cell. Measurement of Em under conditions in which the main background current includes a markedly nonlinear K+ conductance (due to inward rectification) yields complex and somewhat counter-intuitive findings. In fact, there are at least two possible stable values of resting membrane potential for a cell when the nonlinear, inwardly rectifying K+ conductance interacts with the seal current. This type of bistable behavior has been reported in a variety of small mammalian cells, including those from the heart, endothelium, smooth muscle and bone. Our theoretical treatment of these two common experimental situations provides useful mechanistic insights, and suggests practical methods by which these significant limitations, and their impact, can be minimized.
mathematical modeling; inward rectifier; K+ currents; resting potential; seal resistance
The lateral rectus (LR) and medial rectus (MR) extraocular muscles (EOMs) have largely nonoverlapping superior and inferior innervation territories, suggesting functional compartmental specialization. We used magnetic resonance imaging (MRI) in humans to investigate differential compartmental activity in the rectus EOMs during head tilt, which evokes ocular counter-rolling, a torsional vestibulo-ocular reflex (VOR).
MRI in quasi-coronal planes was analyzed during target-controlled central gaze in 90° right and left head tilts in 12 normal adults. Cross sections and posterior partial volumes of the transverse portions of the four rectus EOMs were compared in contiguous image planes 2 mm thick spanning the orbit from origins to globe equator, and used as indicators of contractility.
Horizontal rectus EOMs had significantly greater posterior volumes and maximum cross sections in their inferior compartments (P < 10−8). In orbit tilt up (extorted) compared with orbit tilt down (intorted) head tilts, contractile changes in LR maximum cross section (P < 0.0001) and posterior partial volume (P < 0.05) were significantly greater in the inferior but not in the superior compartment. These changes were not explainable by horizontal or vertical eye position changes. A weaker compartmental effect was suggested for MR. The vertical rectus EOMs did not exhibit significant compartmental contractile changes during head tilt. Mechanical modeling suggests that differential LR contraction may contribute to physiological cyclovertical effects.
Selective activation of the two LR, and possibly MR, compartments correlates with newly recognized segregation of intramuscular innervation into distinct compartments, and probably contributes to noncommutative torsion during the VOR.
Magnetic resonance imaging of extraocular muscles during ocular counter-rolling demonstrates selective activation of the lateral rectus inferior but not superior compartment. This is novel functional evidence that differential rectus compartmental activation contributes to a vestibulo-ocular reflex.
The nuclear hormone receptor PPARγ has been shown to play an immuno-regulatory role in many immune-related cell types and activation of PPARγ has been reported to be an effective therapeutic approach in murine and human autoimmune disease. However, despite an association between lymphopenia and autoimmunity, there have been no prior studies of the role of T cell PPARγ in lymphopenia-associated autoimmunity. In the present studies we examined the role of PPARγ in CD4+ T cells in two murine models of lymphopenia-associated autoimmunity. Surprisingly, we find that PPARγ expression in CD4+ CD25− T cells (Teff) is actually required for development of autoimmunity under lymphopenic conditions. Mechanistically, the inability of PPARγ-deficient (T-PPAR) Teff to mediate lymphopenic autoimmunity is associated with a significant decrease in accumulation of Teff in the spleen, lymph nodes and tissues after adoptive transfer. This abnormal accumulation of T-PPAR Teff is associated with defects in both in vivo proliferation and survival. Additionally, T-PPAR Teff demonstrate decreased cytokine production in inflammatory sites and decreased expression of the homing receptor α4β7. Finally, these abnormalities in T-PPAR Teff function were not elicited by lymphopenia alone, but also required the additional activation involved in the mediation of autoimmunity. Thus, in contrast to its documented immunosuppressive role, we now identify an unexpected function for PPARγ in Teff, namely a role in Teff proliferation and survival in lymphopenia-associated autoimmunity. These findings highlight both the multifunctional role of PPARγ in T cells and the complexity of PPARγ as a potential therapeutic target in autoimmunity.
Reducing neonatal death has been an emerging challenge in low and middle income countries in the past decade. The development of the low cost interventions and their effective delivery are needed to reduce deaths from birth asphyxia. This study will assess the impact of a simplified neonatal resuscitation protocol provided by Helping Babies Breathe (HBB) at a tertiary hospital in Nepal. Perinatal outcomes and performance of skilled birth attendants on management of intrapartum-related neonatal hypoxia will be the main measurements.
The study will be carried out at a tertiary level maternity hospital in Nepal. A prospective cohort-study will include a six-month baseline a six month intervention period and a three-month post intervention period. A quality improvement process cycle will introduce the neonatal resuscitation protocol. A surveillance system, including CCD cameras and pulse oximeters, will be set up to evaluate the intervention.
Along with a technique to improve health workers performance on the protocol, the study will generate evidence on the research gap on the effectiveness of the simplified neonatal resuscitation protocol on intrapartum outcome and early neonatal survival. This will generate a global interest and inform policymaking in relation to delivery care in all income settings.
Global hypoxia-ischemia interrupts oxygen delivery and blood flow to the entire brain. Previous studies of global brain hypoxia ischemia have primarily focused on injury to the cerebral cortex and to the hippocampus. Susceptible neuronal populations also include inhibitory neurons in the thalamic Reticular Nucleus. We therefore investigated the impact of global brain hypoxia-ischemia on the thalamic circuit function in the somatosensory system of young rats. We used single neuron recordings and controlled whisker deflections to examine responses of thalamocortical neurons to sensory stimulation in rat survivors of 9 min of asphyxial cardiac arrest incurred on post-natal day 17. We found that 48–72 hours after cardiac arrest, thalamocortical neurons demonstrate significantly elevated firing rates both during spontaneous activity and in response to whisker deflections. The elevated evoked firing rates persist for at least 6–8 weeks after injury. Despite the overall increase in firing, by 6 weeks, thalamocortical neurons display degraded receptive fields, with decreased responses to adjacent whiskers. Nine min of asphyxial cardiac arrest was associated with extensive degeneration of neurites in the somatosensory nucleus as well as activation of microglia in the Reticular Nucleus. Global brain hypoxia-ischemia during cardiac arrest has a long-term impact on processing and transfer of sensory information by thalamic circuitry. Thalamic circuitry and normalization of its function may represent a distinct therapeutic target after cardiac arrest.
The biochemical cascades associated with cell death after traumatic brain injury (TBI) involve both pro-survival and pro-apoptotic proteins. We hypothesized that elevated cerebrospinal fluid (CSF) Bcl-2 and cytochrome C (CytoC) levels over time would reflect cellular injury response and predict long-term outcomes after TBI. Cerebrospinal fluid Bcl-2 and CytoC levels were measured for 6 days after injury for adults with severe TBI (N=76 subjects; N=277 samples). Group-based trajectory analysis was used to generate distinct temporal biomarker profiles that were compared with Glasgow Outcome Scale (GOS) and Disability Rating Scale (DRS) scores at 6 and 12 months after TBI. Subjects with persistently elevated temporal Bcl-2 and CytoC profiles compared with healthy controls had the worst outcomes at 6 and 12 months (P⩽0.027). Those with CytoC profiles near controls had better long-term outcomes, and those with declining CytoC levels over time had intermediate outcomes. Subjects with Bcl-2 profiles that remained near controls had better outcomes than those with consistently elevated Bcl-2 profiles. However, subjects with Bcl-2 values that started near controls and steadily rose over time had 100% good outcomes by 12 months after TBI. These results show the prognostic value of Bcl-2 and CytoC profiles and suggest a dynamic apoptotic and pro-survival response to TBI.
Bcl-2; biomarker; cerebral spinal fluid; cytochrome C; outcome; traumatic brain injury
Since connective tissue pulleys implement Listing's law by systematically changing rectus extraocular muscle (EOM) pulling directions, non-Listing's law gaze-dependence of the vestibulo-ocular reflex is currently inexplicable. Differential activation of compartments within rectus EOMs may endow the ocular motor system with more behavioral diversity than previously supposed. Innervation to horizontal, but not vertical, rectus EOMs of mammals is segregated into superior and inferior compartments. Magnetic resonance imaging in normal subjects demonstrates contractile changes in the lateral rectus (LR) inferior, but not superior, compartment during ocular counter-rolling (OCR) induced by head tilt. In human orbits ipsilesional to unilateral superior oblique palsy, neither LR compartment exhibits contractile change during head tilt, although the inferior compartment contracts normally in contralesional orbits. This suggests that differential compartmental LR contraction assists normal OCR. Computational simulation suggests that differential compartmental action in horizontal rectus EOMs could achieve more force than required by vertical fusional vergence.
extraocular muscles; magnetic resonance imaging; motor nerve; pulleys; vestibulo-ocular reflex