John A. Melick

Caspase‐3 Mediated Neuronal Death After Traumatic Brain Injury in Rats

Abstract: During programmed cell death, activation of caspase‐3 leads to proteolysis of DNA repair proteins, cytoskeletal proteins, and the inhibitor of caspase‐activated deoxyribonuclease, culminating in morphologic changes and DNA damage defining apoptosis. The participation of caspase‐3 activation in the evolution of neuronal death after traumatic brain injury in rats was examined. Cleavage of pro‐caspase‐3 in cytosolic cellular fractions and an increase in caspase‐3‐like enzyme activity were seen in injured brain versus control. Cleavage of the caspase‐3 substrates DNA‐dependent protein kinase and inhibitor of caspase‐activated deoxyribonuclease and co‐localization of cytosolic caspase‐3 in neurons with evidence of DNA fragmentation were also identified. Intracerebral administration of the caspase‐3 inhibitor N‐benzyloxycarbonyl‐Asp‐Glu‐Val‐Asp‐fluoromethyl ketone (480 ng) after trauma reduced caspase‐3‐like activity and DNA fragmentation in injured brain versus vehicle at 24 h. Treatment with N‐benzyloxycarbonyl‐Asp‐Glu‐Val‐Asp‐fluoromethyl ketone for 72 h (480 ng/day) reduced contusion size and ipsilateral dorsal hippocampal tissue loss at 3 weeks but had no effect on functional outcome versus vehicle. These data demonstrate that caspase‐3 activation contributes to brain tissue loss and downstream biochemical events that execute programmed cell death after traumatic brain injury. Caspase inhibition may prove efficacious in the treatment of certain types of brain injury where programmed cell death occurs.

Cerebral perfusion during anesthesia with fentanyl, isoflurane, or pentobarbital in normal rats studied by arterial spin-labeled MRI

The influence of anesthetic agents on cerebral blood flow (CBF) was tested in normal rats. CBF is quantified with arterial spin‐labeled MRI in rats anesthetized with either an opiate (fentanyl), a potent inhalation anesthetic agent (isoflurane), or a barbiturate (pentobarbital) using doses commonly employed in experimental paradigms. CBF values were found to be about 2.5–3 times lower in most regions analyzed during anesthesia with either fentanyl (with N2O/O2) or pentobarbital vs. isoflurane (with N2O/O2), in agreement with findings utilizing invasive measurement techniques. CBF was heterogeneous in rats anesthetized with isoflurane (with N2O/O2), but relatively homogeneous in rats anesthetized with either fentanyl (with N2O/O2) or pentobarbital, also in agreement with studies using other techniques. Magn Reson Med 46:202–206, 2001.

A dual role for poly-ADP-ribosylation in spatial memory acquisition after traumatic brain injury in mice involving NAD+ depletion and ribosylation of 14-3-3γ

Poly(ADP‐ribose) polymerase‐1 (PARP‐1) is a homeostatic enzyme that paradoxically contributes to disturbances in spatial memory acquisition after traumatic brain injury (TBI) in transgenic mice, thought to be related to depletion of its substrate nicotinamide adenine dinucleotide (NAD+). In this study, systemic administration of the PARP‐1 inhibitor 5‐iodo‐6‐amino‐1,2‐benzopyrone (INH2BP) after TBI preserved brain NAD+ levels and dose‐dependently reduced poly‐ADP‐ribosylation 24 h after injury. While moderate‐dose INH2BP improved spatial memory acquisition after TBI; strikingly, both injured‐ and sham‐mice receiving high‐dose INH2BP were unable to learn in the Morris‐water maze. Poly‐ADP‐ribosylated peptides identified using a proteomics approach yielded several proteins potentially associated with memory, including structural proteins (tubulin α and β, γ‐actin, and α‐internexin neuronal intermediate filament protein) and 14‐3‐3γ. Nuclear poly‐ADP‐ribosylation of 14‐3‐3γ was completely inhibited by the dose of INH2BP that produced profound memory disturbances. Thus, partial inhibition of poly‐ADP‐ribosylation preserves NAD+ and improves functional outcome after TBI, whereas more complete inhibition impairs spatial memory acquisition independent of injury, and is associated with ribosylation of 14‐3‐3γ.

Suppression of cerebral metabolic rate for oxygen (CMRO2) by mild hypothermia compared with thiopental.

If the efficacy of hypothermia and barbiturates in ameliorating ischemic brain injury lies in reducing the cerebral metabolic rate of oxygen (CMRO2), the greater efficacy of mild hypothermia (34 degrees C) compared with barbiturates is inconsistent with the 15-20% reduction of CMRO2 caused by mild hypothermia compared with 50% caused by barbiturates. This paradox, we hypothesized, derives from the fact that whereas barbiturates lower CMRO2 associated with EEG activity or thiopental (TP)-suppressible CMRO2, not essential for cellular viability, hypothermia lowers CMRO2 associated with providing energy, i.e., adenosine triphosphate, to maintain transmembrane ion gradients or TP-nonsuppressible CMRO2, essential for neuronal viability. To test this hypothesis, we measured whole brain cerebral blood flow (CBF) and CMRO2 in two groups of rats mechanically ventilated with 70% N2O/30% O2 before and after TP-induced isoelectric EEG. In the normothermic group (n = 7), measurements were made at a brain temperature (Tb) of 38 degrees C, while in the hypothermic group (n = 7), they were made at 34 degrees C. In the normothermic group, TP-induced isoelectric EEG reduced CMRO2 by 50%, from 7.92 +/- 1.05 to 3.95 +/- 0.70 ml 100 g-1 min-1 (mean +/- = SD). Thus, at 38 degrees C, TP-suppressible and TP-nonsuppressible CMRO2 were both 50 +/- 4% of total CMRO2. In the hypothermic group, decreasing Tb from 38 to 34 degrees C caused a 17% decline in CMRO2, from 7.62 +/- 1.92 to 6.28 +/- 1.22 ml 100 g-1 min-1 (p > 0.05). AT 34 degrees C, TP infusion lowered CMRO2 to 2.15 = 0.46 ml 100 g-1 min-1. At 34 degrees C, TP-suppressible and TP-nonsuppressible CMRO2 values were 64 +/- 7% and 36 +/- 8% of total CMRO2, respectively. TP lowered CBF by 50% at both 38 and 34 degrees C. In conclusion, mild hypothermia selectively lowers TP-nonsuppressible CMRO2 associated with the maintenance of viability rather than EEG-associated or TP-suppressible CMRO2.

Cerebrovascular and cerebrometabolic effects of intracarotid infused platelet-activating factor in rats

Platelet-activating factor has been implicated in a variety of disease processes including ischemic brain injury and endotoxic shock, but its effects on cerebral blood flow (CBF) and metabolism in normal brain have not been described. The effects of platelet-activating factor on global CBF (hydrogen clearance) and the global cerebral metabolic rate for oxygen (CMRO2) were studied in halothane-N2O anesthetized Wistar rats. Hexadecyl-platelet-activating factor infused into the right carotid artery (67 pmol/min) for 60 min decreased mean arterial pressure (MAP) from 122 ±4 (x ± SEM) to 77 ± 6 mm Hg and CBF from 159 ± 12 to 116 ± 14 ml/100 g/min (p < 0.002). In contrast, CMRO2 increased from 9.7 ± 0.9 to 11.7 ± 1.1 ml/100 g/min after 15 min (p < 0.05). In controls rendered similarly hypotensive by blood withdrawal and infused with the platelet-activating factor vehicle, CMRO2 was unchanged, whereas CBF transiently decreased then returned to baseline at 60 min. These cerebrovascular and cerebrometabolic effects of PAF are reminiscent of and may be relevant to hypoperfusion and hypermetabolism observed after global brain ischemia and in endotoxic shock.

Effect of neutropenia and granulocyte colony stimulating factor-induced neutrophilia on blood-brain barrier permeability and brain edema after traumatic brain injury in rats.

ObjectiveGranulocyte colony stimulating factor (GCSF) has been used to increase systemic absolute neutrophil count (ANC) in patients with severe traumatic brain injury to reduce nosocomial infection risk. However, the effect of increasing systemic ANC on the pathogenesis of experimental traumatic brain injury has not been studied. Thus, we evaluated the effect of systemic ANC on blood-brain barrier (BBB) damage and brain edema after traumatic brain injury in rats. DesignExperimental study. SettingResearch laboratory at the University of Pittsburgh, PA. SubjectsForty-three adult male Sprague-Dawley rats. InterventionsProtocol I: rats were randomized to receive either vinblastine sulfate to reduce ANC, GCSF to increase ANC, or saline before controlled cortical impact (CCI) of moderate overall severity. Evans blue was used to assess BBB damage at 4–24 hrs after CCI. Protocol II: rats received GCSF or saline before CCI. Brain edema was estimated at 24 hrs using (wet − dry) ÷ wet weight method. Protocol III: rats received GCSF or saline before CCI. Brain neutrophil accumulation was estimated at 24 hrs using a myeloperoxidase assay. Measurements and Main ResultsPhysiologic variables were controlled before CCI was maintained at normal in all animals before traumatic brain injury. No rats were anemic, hypoglycemic, or hypotensive before CCI. Protocol I: compared with control, systemic ANC decreased in vinblastine-treated rats and increased in GCSF-treated rats. BBB damage correlated with systemic ANC. Protocol II: mean systemic ANC before traumatic brain injury increased 15-fold in rats given GCSF vs. control; however no difference in brain edema was observed at 24 hrs after injury between groups. Protocol III: median systemic ANC at the time of CCI was increased ten-fold in rats given GCSF vs. control. No difference in brain myeloperoxidase activity 24 hrs after CCI was observed in rats treated with GCSF vs. control. ConclusionsSystemic ANC influences BBB damage after traumatic brain injury produced by CCI. Because BBB damage and brain edema are discordant, mechanisms other than BBB damage likely predominate in the pathogenesis of brain edema after contusion. The implications of increased BBB permeability with the administration of GCSF in our model remains to be determined. Increasing systemic ANC before CCI with GCSF administration does not increase posttraumatic brain neutrophil accumulation or brain edema after CCI in rats. The finding that neutrophil infiltration is not enhanced by systemic neutrophilia suggests that the ability of GCSF-stimulated neutrophils to migrate into injured tissue may be impaired. Further studies are needed to evaluate the effects of GCSF administration on secondary injury and functional outcome in experimental models of traumatic brain injury.

Magnetic Resonance Imaging Assessment of Macrophage Accumulation in Mouse Brain after Experimental Traumatic Brain Injury

Macrophages contribute to secondary damage and repair after central nervous system (CNS) injury. Micron-sized paramagnetic iron oxide (MPIO) particles can label macrophages in situ, facilitating three-dimensional (3D) mapping of macrophage accumulation following traumatic brain injury (TBI), via ex vivo magnetic resonance microscopy (MRM) and in vivo monitoring with magnetic resonance imaging (MRI). MPIO particles were injected intravenously (iv; 4.5 mg Fe/Kg) in male C57BL/6J mice (n = 21). A controlled cortical impact (CCI) was delivered to the left parietal cortex. Five protocols were used in naive and injured mice to assess feasibility, specificity, and optimal labeling time. In vivo imaging was carried out at 4.7 Tesla (T). Brains were then excised for 3D MRM at 11.7 T. Triple-label immunofluorescence (MPIO via Dragon Green, macrophages via F480, and nuclei via 4,6-diamidino-2-phenylindole [DAPI]) of brain sections confirmed MPIO particles within macrophages. MRM of naives showed an even distribution of a small number of MPIO-labeled macrophages in the brain. MRM at 48-72 h after CCI and MPIO injection revealed MPIO-labeled macrophages accumulated in the trauma region. When MPIO particles were injected 6 days before CCI, MRM 48 h after CCI also revealed labeled cells at the injury site. In vivo studies of macrophage accumulation by MRI suggest that this approach is feasible, but requires additional optimization. We conclude that MPIO labeling and ex vivo MRM mapping of macrophage accumulation for assessment of TBI is readily accomplished. This new technique could serve as an adjunct to conventional MR approaches by defining inflammatory mechanisms and therapeutic efficacy of anti-inflammatory agents in experimental TBI.

Administration of adenosine receptor agonists or antagonists after controlled cortical impact in mice: effects on function and histopathology

Adenosine is an endogenous neuroprotectant via anti-excitotoxic effects at A(1) receptors, and blood flow promoting and anti-inflammatory effects at A(2a) receptors. Previous studies showed improved motor function after fluid percussion injury (FPI) in rats treated with the broad-spectrum adenosine receptor agonist 2-chloroadenosine (2-CA). We studied the effects of 2-CA, a specific A(1) agonist (2-chloro-N(6)-cyclopentyladenosine, CCPA), and a specific A(1) antagonist (8-cyclopentyl-1,3-dipropylxanthine, DPCPX) on motor task and Morris water maze (MWM) performance, and histopathology (contusion volume, hippocampal cell counts) after controlled cortical impact (CCI) in mice. Each agent (12 nmol), or respective vehicle (saline or DMSO) was injected into dorsal hippocampus beneath the contusion immediately after CCI or craniotomy (sham). 2-CA treatment attenuated wire grip deficits after CCI (P<0.05 versus other treatments). DPCPX treatment exacerbated deficits on beam balance (P<0.05 versus sham). No treatment effect was seen on MWM performance, although there was a deleterious effect of the DMSO vehicle used for DPCPX. Contusion volume tended to be attenuated by 2-CA (P=0.08 versus saline) and increased after either DMSO or DPCPX (P<0.05 versus all groups). CA1 and CA3 counts were decreased in all groups versus sham. However, treatment with the selective A(1) agonist CCPA attenuated the CA3 cell loss (P<0.05 versus other treatment). We suggest that the beneficial effect of the broad spectrum adenosine receptor agonist 2-CA on motor function after CCI is not mediated solely by effects at the A(1) receptor.

Mild Hypothermia Decreases Fentanyl and Midazolam Steady-State clearance in a Rat Model of Cardiac Arrest

Objectives:Therapeutic hypothermia is widely employed for neuroprotection after cardiac arrest. However, concern regarding elevated drug concentrations during hypothermia and increased adverse drug reaction risk complicates concurrent pharmacotherapy. Many commonly used medications in critically ill patients rely on the cytochrome P450 3A isoform for their elimination. Therefore, our study objectives were to determine the effect of mild hypothermia on the in vivo pharmacokinetics of fentanyl and midazolam, two clinically relevant cytochrome P450 3A substrates, after cardiac arrest and to investigate the mechanisms of these alterations. Design:Prospective, randomized, controlled study. Setting:University research laboratory. Subjects:Thirty-two adult male Sprague-Dawley rats. Interventions:An asphyxial cardiac arrest rat model was used and mild hypothermia (33°C) was induced 1 hr post injury by surface cooling and continued for 10 hrs to mimic the prolonged clinical application of hypothermia accompanied by intensive care interventions. Fentanyl and midazolam were independently administered by intravenous infusion and plasma and brain concentrations were analyzed using ultraperformance liquid chromatography tandem mass spectrometry. Cytochrome P450 3a2 protein expression was measured and a Michaelis-Menten enzyme kinetic analysis was performed at 37°C and 33°C using control rat microsomes. Measurements and Main Results:Mild hypothermia decreased the systemic clearance of both fentanyl (61.5 ± 11.5 to 48.9 ± 8.95 mL/min/kg; p < .05) and midazolam (89.2 ± 12.5 to 73.6 ± 12.1 mL/min/kg; p < .05) after cardiac arrest. The elevated systemic concentrations did not lead to parallel increased brain exposures of either drug. Mechanistically, no differences in cytochrome P450 3a2 expression was observed, but the in vitro metabolism of both drugs was decreased at 33°C vs. 37°C through reductions in enzyme metabolic capacity rather than substrate affinity. Conclusions:Mild hypothermia reduces the systemic clearances of fentanyl and midazolam in rats after cardiac arrest through alterations in cytochrome P450 3a2 metabolic capacity rather than enzyme affinity as observed with other cytochrome P450s. Contrasting effects on blood and brain levels further complicates drug dosing. Consideration of the impact of hypothermia on medications whose clearance is dependent on P450 3A metabolism is warranted. (Crit Care Med 2012; 40:–1228)

boc-Aspartyl(OMe)-fluoromethylketone attenuates mitochondrial release of cytochrome c and delays brain tissue loss after traumatic brain injury in rats

The pathobiology of traumatic brain injury (TBI) includes activation of multiple caspases followed by cell death with a spectrum of apoptotic phenotypes. There are initiator (e.g. caspase-2, −8, and −9) and effector (e.g. caspase-3 and −7) caspases. Recently, caspase-2 and −8 have been shown to regulate cell death via provoking cytochrome c release from the mitochondria upstream of caspase-9. Here, we show that an intracerebral injection of the pan-caspase inhibitor boc-Aspartyl(OMe)-fluoromethylketone (BAF; 1 μmol) 1 min after TBI in rats reduces caspase-3-like activity, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) and tissue damage, and cytochrome c release in ipsilateral cortex at 24 h versus vehicle. To investigate whether either caspase-2 and/or caspase-8 activation may contribute to cytochrome release, the effect of BAF treatment on caspase-2 and caspase-8 proteolysis was also examined. boc-aspartyl(OMe)-fluoromethylketone treatment inhibited proteolysis of caspase-2 but not caspase-8 24 h after TBI in rats versus vehicle. However, BAF with or without nerve growth factor (12.5 ng/hx14 days intracerebrally via osmotic pump) did not result in differences in motor function, Morris water maze performance, hippocampal neuron survival, nor contusion volume at 14 days. These data suggest that BAF treatment reduces acute cell death after TBI by inhibiting mitochondrial release of cytochrome c, possibly via a mechanism involving initiator caspases; however, BAF appears to delay cell death, rather than result in permanent protection.