MRI assessment of cerebral blood flow after experimental traumatic brain injury combined with hemorrhagic shock in mice.

Secondary insults such as hypotension or hemorrhagic shock (HS) can greatly worsen outcome after traumatic brain injury (TBI). We recently developed a mouse combined injury model of TBI and HS using a controlled cortical impact (CCI) model and showed that 90 minutes of HS can exacerbate neuronal death in hippocampus beneath the contusion. This combined injury model has three clinically relevant phases, a shock, pre hospital, and definitive care phases. Mice were randomly assigned to four groups, shams as well as a CCI only, an HS only, and a CCI+HS groups. The CCI and HS reduced cerebral blood flow (CBF) in multiple regions of interest (ROIs) in the hemisphere ipsilateral and contralateral to injury. Hemorrhagic shock to a level of ∼30 mm Hg exacerbated the CCI-induced CBF reductions in multiple ROIs ipsilateral to injury (hemisphere and thalamus) and in the hemisphere contralateral to injury (hemisphere, thalamus, hippocampus, and cortex, all P<0.05 versus CCI only, HS only or both). An important effect of HS duration was also seen after CCI with maximal CBF reduction seen at 90 minutes (P<0.0001 group-time effect in ipsilateral hippocampus). Given that neuronal death in hippocampus is exacerbated by 90 minutes of HS in this model, our data suggest an important role for exacerbation of posttraumatic ischemia in mediating the secondary injury in CCI plus HS. In conclusion, the serial, non invasive assessment of CBF using ASL-MRI (magnetic resonance imaging with arterial spin labeling) is feasible in mice even in the complex setting of combined CCI+HS. The impact of resuscitation therapies and various mutant mouse strains on CBF and other outcomes merits investigation in this model.

Automated detection and characterization of SPIO-labeled cells and capsules using magnetic field perturbations.

Understanding how individual cells behave inside living systems will help enable new diagnostic tools and cellular therapies. Superparamagnetic iron oxide particles can be used to label cells and theranostic capsules for noninvasive tracking using MRI. Contrast changes from superparamagnetic iron oxide are often subtle relative to intrinsic sources of contrast, presenting a detection challenge. Here, we describe a versatile postprocessing method, called Phase map cross-correlation Detection and Quantification (PDQ), that automatically identifies localized deposits of superparamagnetic iron oxide, estimating their volume magnetic susceptibility and magnetic moment. To demonstrate applicability, PDQ was used to detect and characterize superparamagnetic iron oxide-labeled magnetocapsules implanted in porcine liver and suspended in agarose gel. PDQ was also applied to mouse brains infiltrated by MPIO-labeled macrophages following traumatic brain injury; longitudinal, in vivo studies tracked individual MPIO clusters over 3 days, and tracked clusters were corroborated in ex vivo brain scans. Additionally, we applied PDQ to rat hearts infiltrated by MPIO-labeled macrophages in a transplant model of organ rejection. PDQ magnetic measurements were signal-to-noise ratio invariant for images with signal-to-noise ratio > 11. PDQ can be used with conventional gradient-echo pulse sequences, requiring no extra scan time. The method is useful for visualizing biodistribution of cells and theranostic magnetocapsules and for measuring their relative iron content.

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.

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.

Effect of inducible nitric oxide synthase on cerebral blood flow after experimental traumatic brain injury in mice.

Inducible nitric oxide synthase (iNOS) has been suggested to play a complex role in the response to central nervous system insults such as traumatic brain injury (TBI) and cerebral ischemia. In the current study, we quantified maps of regional cerebral blood flow (CBF) using an arterial spin-labeling magnetic resonance imaging (MRI) technique, at 24 and 72 h after experimental TBI in iNOS knockout (KO) and wild-type (WT) mice. Our hypothesis was that iNOS would contribute to the level of CBF at 72 h after experimental TBI in mice. Comparing anatomical brain regions of interest (ROIs) at 24-h post controlled cortical impact (CCI), there were significant reductions in CBF in the hemisphere, cortex, and contusion-rich area of the cortex of injured animals versus naive, regardless of genotype. Regional assessment of CBF at 72 h after injury demonstrated that recovery of CBF was reduced in the ipsilateral hippocampus, thalamus, and amygdala/piriform cortex in iNOS KO versus WT mice by 26%, 15%, and 21%, respectively; this attenuated recovery was restricted to structures outside the contusion. These regions with reduced CBF in iNOS KO mice represented ROIs where CBF in the WT was either numerically or statistically greater than that seen in respective WT naive, suggesting a contribution of iNOS to delayed posttraumatic hyperemia. However, pixel analysis denoted that the contribution of iNOS to CBF at 72 h was not limited to hyperemia flows. In conclusion, iNOS plays a role in the recovery of CBF after CCI in mice. Questions remain if this effect represents a homeostatic component of CBF recovery, pathologic vasodilatation linked to inflammation, or NO-mediated facilitation of angiogenesis.

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 micromol) 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/h x 14 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.

Murine orthostatic response during prolonged vertical studies: effect on cerebral blood flow measured by arterial spin-labeled MRI.

High-field MRI scanners are, in principle, well suited for mouse studies; however, many high-field magnets employ a vertical design that may influence the physiological state of the rodent. The purpose of this study was to investigate the orthostatic response of cerebral blood flow (CBF) in mice during a prolonged MR experiment in the vertical position. Arterial spin-labeled (ASL) MRI was performed at 4.7-Tesla with a 15-cm gradient insert that allowed horizontal and vertical CBF measurements to be obtained with the same scanner. For mice in the head-up (HU) vertical position, CBF decreased by approximately 40% compared to the horizontal position, although blood pressure did not differ. Furthermore, CBF values for vertically positioned mice treated with phenylephrine remained constant while blood pressure increased. These results support the conclusion that cerebral autoregulation was intact, albeit at a lower level. Since CBF recovers to near horizontal values by volume loading with saline, it appears that a decrease in central venous pressure (CVP) leading to an increase in sympathetic tone may be a contributing mechanism for lowered CBF. This suggests that using an HU vertical position for MRI in mice may have broader implications, especially for studies that rely on CBF (such as BOLD and fMRI).

Characterization of the effects of adenosine receptor agonists on cerebral blood flow in uninjured and traumatically injured rat brain using continuous arterial spin-labeled magnetic resonance imaging.

Hypoperfusion after traumatic brain injury may exacerbate damage. Adenosine, a vasodilator, regulates cerebral blood flow (CBF). Treatment with adenosine receptor agonists has shown benefit in experimental CNS trauma; however, their effects on CBF after injury remain undefined. We used magnetic resonance imaging to assess CBF in uninjured rats both early and at 24 h after intrahippocampal administration of either the nonselective adenosine receptor agonist 2-chloroadenosine (2-CA, 12 nmol) or the A(2A)-receptor agonist 2-p-(2-carboxyethyl)-phenethylamino-5'-N-ethylcarbox-amidoadenosine (CGS 21680, 6 nmol). We also assessed the effects of these agents on cerebral metabolic rate for glucose (CMRglu). We then assessed the effect of 2-CA on CBF at 3.5 to 5 h after controlled cortical impact (CCI). Injection of 2-CA into uninjured rat brain produced marked increases in CBF in ipsilateral hippocampus and cortex versus vehicle (P<0.05); CBF increases persisted even at 24 h. Measurement of hippocampal levels of 2-CA showed persistent increases to 24 h. CGS 21680 produced even more marked global increases in CBF than seen with 2-CA (2-6-fold versus vehicle, P<0.05 in 10/12 regions of interest (ROIs)). Neither agonist altered CMRglu versus vehicle. After CCI, 2-CA increased CBF in ipsilateral hippocampal and hemispheric ROIs (P<0.05 versus vehicle), but the response was attenuated at severe injury levels. We report marked increases in CBF after injection of adenosine receptor agonists into uninjured rat brain despite unaltered CMRglu. 2-Chloroadenosine produced enduring increases in CBF in uninjured brain and attenuated posttraumatic hypoperfusion. Future studies of adenosine-related therapies in CNS injury should address the role of CBF.

Hyperglycolysis is exacerbated after traumatic brain injury with fentanyl vs. isoflurane anesthesia in rats.

Despite common use of narcotics in the clinical management of severe traumatic brain injury (TBI), in experimental models rats treated with fentanyl have exhibited worse functional outcome and more CA1 hippocampal death than rats treated with standard isoflurane anesthesia. We hypothesized that greater post-traumatic excitotoxicity, reflected by cerebral glucose utilization (CMRglu), may account for detrimental effects of fentanyl vs. isoflurane. Rats were anesthetized with either isoflurane (1% by inhalation) or fentanyl (10 mcg/kg iv bolus then 50 mcg/kg/h infusion). 14C-deoxyglucose autoradiography was performed 45 min after controlled cortical impact (CCI) to left parietal cortex (n=4 per anesthetic group) or in uninjured rats after 45 min of anesthesia (n=3 per anesthetic group). Uninjured rats treated with fentanyl vs. isoflurane showed 35-45% higher CMRglu in all brain structures (p<0.05) except CA3. After TBI in rats treated with isoflurane, CMRglu increased significantly only in ipsilateral CA1 and ipsilateral parietal cortex (p<0.05 vs. isoflurane uninjured). Conversely, after TBI in rats treated with fentanyl, CMRglu increased markedly and bilaterally in CA1 and CA3 (p<0.05 vs. fentanyl uninjured), but not ipsilateral parietal cortex. In contralateral CA1, CMRglu was nearly two times greater after TBI in fentanyl vs. isoflurane treated rats (p<0.05). Hyperglycolysis was exacerbated in CA1 and CA3 hippocampus after TBI in rats treated with fentanyl vs. isoflurane anesthesia. This post-traumatic hyperglycolysis suggests greater excitotoxicity and concurs with reports of worse functional outcome and more CA1 hippocampal death after TBI with fentanyl vs. isoflurane anesthesia.

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.