Inflammatory cytokine receptor blockade in a rodent model of mild traumatic brain injury.

In rodent models of traumatic brain injury (TBI), both Interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNFα) levels increase early after injury to return later to basal levels. We have developed and characterized a rat mild fluid percussion model of TBI (mLFP injury) that results in righting reflex response times (RRRTs) that are less than those characteristic of moderate to severe LFP injury and yet increase IL-1α/ß and TNFα levels. Here we report that blockade of IL-1α/ß and TNFα binding to IL-1R and TNFR1, respectively, reduced neuropathology in parietal cortex, hippocampus, and thalamus and improved outcome. IL-1ß binding to the type I IL-1 receptor (IL-1R1) can be blocked by a recombinant form of the endogenous IL-1R antagonist IL-1Ra (Kineret). TNFα binding to the TNF receptor (TNFR) can be blocked by the recombinant fusion protein etanercept, made up of a TNFR2 peptide fused to an Fc portion of human IgG1. There was no benefit from the combined blockades compared with individual blockades or after repeated treatments for 11 days after injury compared with one treatment at 1 hr after injury, when measured at 6 hr or 18 days, based on changes in neuropathology. There was also no further enhancement of blockade benefits after 18 days. Given that both Kineret and etanercept given singly or in combination showed similar beneficial effects and that TNFα also has a gliotransmitter role regulating AMPA receptor traffic, thus confounding effects of a TNFα blockade, we chose to focus on a single treatment with Kineret.

A rodent model of mild traumatic brain blast injury.

One of the criteria defining mild traumatic brain injury (mTBI) in humans is a loss of consciousness lasting for less than 30 min. mTBI can result in long-term impairment of cognition and behavior. In rats, the length of time it takes a rat to right itself after injury is considered to be an analog for human return to consciousness. This study characterized a rat mild brain blast injury (mBBI) model defined by a righting response reflex time (RRRT) of more than 4 min but less than 10 min. Assessments of motor coordination relying on beam-balance and foot-fault assays and reference memory showed significant impairment in animals exposed to mBBI. This study's hypothesis is that there are inflammatory outcomes to mTBI over time that cause its deleterious effects. For example, mBBI significantly increased brain levels of interleukin (IL)-1ß and tumor necrosis factor-α (TNFα) protein. There were significant inflammatory responses in the cortex, hippocampus, thalamus, and amygdala 6 hr after mBBI, as evidenced by increased levels of the inflammatory markers associated with activation of microglia and macrophages, ionized calcium binding adaptor 1 (IBA1), impairment of the blood-brain barrier, and significant neuronal losses. There were significant increases in phosphorylated Tau (p-Tau) levels, a putative precursor to the development of neuroencephalopathy, as early as 6 hr after mBBI in the cortex and the hippocampus but not in the thalamus or the amygdala. There was an apparent correlation between RRRTs and p-Tau protein levels but not IBA1. These results suggest potential therapies for mild blast injuries via blockade of the IL-1ß and TNFα receptors.

Optoacoustic monitoring of cerebral venous blood oxygenation though intact scalp in large animals.

Monitoring (currently invasive) of cerebral venous blood oxygenation is a key to avoiding hypoxia-induced brain injury resulting in death or severe disability. Noninvasive, optoacoustic monitoring of cerebral venous blood oxygenation can potentially replace existing invasive methods. To the best of our knowledge, we report for the first time noninvasive monitoring of cerebral venous blood oxygenation through intact scalp that was validated with invasive, "gold standard" measurements. We performed an in vivo study in the sheep superior sagittal sinus (SSS), a large midline cerebral vein, using our novel, multi-wavelength optoacoustic system. The study results demonstrated that: 1) the optoacoustic signal from the sheep SSS is detectable through the thick, intact scalp and skull; 2) the SSS signal amplitude correlated well with wavelength and actual SSS blood oxygenation measured invasively using SSS catheterization, blood sampling, and measurement with "gold standard" CO-Oximeter; 3) the optoacoustically predicted oxygenation strongly correlated with that measured with the CO-Oximeter. Our results indicate that monitoring of cerebral venous blood oxygenation may be performed in humans noninvasively and accurately through the intact scalp using optoacoustic systems because the sheep scalp and skull thickness is comparable to that of humans whereas the sheep SSS is much smaller than that of humans.

Optoacoustic monitoring of cerebral venous blood oxygenation through extracerebral blood.

There is strong clinical evidence that controlling cerebral venous oxygenation (oxyhemoglobin saturation) is critically important for patients with severe traumatic brain injury as well as for patients undergoing cardiac surgery. However, the only available method for cerebral venous blood oxygenation monitoring is invasive and requires catheterization of the internal jugular vein. We designed and built a novel optoacoustic monitor of cerebral venous oxygenation as measured in the superior sagittal sinus (SSS), the large midline cerebral vein. To the best of our knowledge, optical monitoring of cerebral venous blood oxygenation through overlying extracerebral blood is reported for the first time in this paper. The system was capable of detecting SSS signals in vivo at 700, 800, and 1064 nm through the thick (5-6 mm) sheep skull containing the circulating blood. The high (submillimeter) in-depth resolution of the system provided identification of the SSS peaks in the optoacoustic signals. The SSS peak amplitude closely followed the actual SSS blood oxygenation measured invasively using catheterization, blood sampling, and "gold standard" CO-Oximetry. Our data indicate the system may provide accurate measurement of the SSS blood oxygenation in patients with extracerebral blood over the SSS.

Noninvasive monitoring of cerebral blood oxygenation in ovine superior sagittal sinus with novel multi-wavelength optoacoustic system.

Noninvasive monitoring of cerebral blood oxygenation with an optoacoustic technique offers advantages over current invasive and noninvasive methods. We report the results of in vivo studies in the sheep superior sagittal sinus (SSS), a large central cerebral vein. We changed blood oxygenation by increasing and decreasing the inspired fraction of oxygen (FiO(2)). Optoacoustic measurements from the SSS were performed at wavelengths of 700, 800, and 1064 nm using an optical parametric oscillator as a source of pulsed near-infrared light. Actual oxygenation of SSS blood was measured with a CO-Oximeter in blood samples drawn from the SSS through a small craniotomy. The amplitude of the optoacoustic signal induced in the SSS blood at lambda = 1064 nm closely followed the changes in blood oxygenation, at lambda = 800 nm was almost constant, and at lambda = 700 nm was changing in the opposite direction, all in accordance with the absorption spectra of oxy- and deoxyhemoglobin. The optoacoustically predicted oxygenation correlated well with actual blood oxygenation in sheep SSS (R(2) = 0.965 to 0.990). The accuracy was excellent, with a mean difference of 4.8% to 9.3% and a standard deviation of 2.8% to 4.2%. To the best of our knowledge, this paper reports for the first time accurate measurements of cerebral venous blood oxygenation validated against the "gold standard" CO-Oximetry method.

In vivo monitoring of blood oxygenation in large veins with a triple-wavelength optoacoustic system.

A noninvasive optoacoustic technique could be a clinically useful alternative to existing, invasive methods for cerebral oxygenation monitoring. Recently we proposed to use an optoacoustic technique for monitoring cerebral blood oxygenation by probing large cerebral and neck veins including the superior sagittal sinus and the internal jugular vein. In these studies we used a multi-wavelength optoacoustic system with a nanosecond optical parametric oscillator as a light source and a custom-made optoacoustic probe for the measurement of the optoacoustic signals in vivo from the area of the sheep neck overlying the external jugular vein, which is similar in diameter and depth to the human internal jugular vein. Optoacoustic signals induced in venous blood were measured with high resolution despite the presence of a thick layer of tissues (up to 10 mm) between the external jugular vein and the optoacoustic probe. Three wavelengths were chosen to provide accurate and stable measurements of blood oxygenation: signals at 700 nm and 1064 nm demonstrated high correlation with actual oxygenation measured invasively with CO-Oximeter ("gold standard"), while the signal at 800 nm (isosbestic point) was independent of blood oxygenation and was used for calibration.

Concentrations of extracellular free zinc (pZn)e in the central nervous system during simple anesthetization, ischemia and reperfusion.

"Free Zn2+" (rapidly exchangeable Zn2+) is stored along with glutamate in the presynaptic terminals of specific specialized (gluzinergic) cerebrocortical neurons. This synaptically releasable Zn2+ has been recognized as a potent modulator of glutamatergic transmission and as a key toxin in excitotoxic neuronal injury. Surprisingly (despite abundant work on bound zinc), neither the baseline concentration of free Zn2+ in the brain nor the presumed co-release of free Zn2+ and glutamate has ever been directly observed in the intact brain in vivo. Here, we show for the first time in dialysates of rat and rabbit brain and human CSF samples from lumbar punctures that: (i) the resting or "tonic" level of free Zn2+ signal in the extracellular fluid of the rat, rabbit and human being is approximately 19 nM (95% range: 5-25 nM). This concentration is 15,000-fold lower than the "300 microM" concentration which is often used as the "physiological" concentration of free zinc for stimulating neural tissue. (ii) During ischemia and reperfusion in the rabbit, free zinc and glutamate are (as has often been presumed) released together into the extracellular fluid. (iii) Unexpectedly, Zn2+ is also released alone (without glutamate) at a variable concentration for several hours during the reperfusion aftermath following ischemia. The source(s) of this latter prolonged release of Zn2+ is/are presumed to be non-synaptic and is/are now under investigation. We conclude that both Zn2+ and glutamate signaling occur in excitotoxicity, perhaps by two (or more) different release mechanisms.

Analysis of long-term gene expression in neurons of the hippocampal subfields following traumatic brain injury in rats.

After experimental traumatic brain injury (TBI), widespread neuronal loss is progressive and continues in selectively vulnerable brain regions, such as the hippocampus, for months to years after the initial insult. To clarify the molecular mechanisms underlying secondary or delayed cell death in hippocampal neurons after TBI, we compared long-term changes in gene expression in the CA1, CA3 and dentate gyrus (DG) subfields of the rat hippocampus at 24 h and 3, 6, and 12 months after TBI with changes in gene expression in sham-operated rats. We used laser capture microdissection to collect several hundred hippocampal neurons from the CA1, CA3, and DG subfields and linearly amplified the nanogram samples of neuronal RNA with T7 RNA polymerase. Subsequent quantitative analysis of gene expression using ribonuclease protection assay revealed that mRNA expression of the anti-apoptotic gene, Bcl-2, and the chaperone heat shock protein 70 was significantly downregulated at 3, 6 (Bcl-2 only), and 12 months after TBI. Interestingly, the expression of the pro-apoptotic genes caspase-3 and caspase-9 was also significantly decreased at 3, 6 (caspase-9 only), and 12 months after TBI, suggesting that long-term neuronal loss after TBI is not mediated by increased expression of pro-apoptotic genes. The expression of two aging-related genes, p21 and integrin beta3 (ITbeta3), transiently increased 24 h after TBI, returned to baseline levels at 3 months and significantly decreased below sham levels at 12 months (ITbeta3 only). Expression of the gene for the antioxidant glutathione peroxidase-1 also significantly increased 6 months after TBI. These results suggest that decreased levels of neuroprotective genes may contribute to long-term neurodegeneration in animals and human patients after TBI. Conversely, long-term increases in antioxidant gene expression after TBI may be an endogenous neuroprotective response that compensates for the decrease in expression of other neuroprotective genes.

Accurate, noninvasive measurement of total hemoglobin concentration with optoacoustic technique.

The measurement of total hemoglobin concentration is currently invasive and time consuming. The optoacoustic technique may provide accurate and noninvasive measurements of total hemoglobin concentration by probing blood vessels. We studied the influence of blood vessel diameter and lateral displacement of the optoacoustic probe on accuracy of total hemoglobin concentration measurements with this technique.

In vivo noninvasive monitoring of cerebral blood with optoacoustic technique.

We present the results of blood oxygenation (oxyhemoglobin saturation) measurements using an optoacoustic system in vivo in the superior sagittal sinus of sheep. The system included a nanosecond Nd:YAG laser as a source of radiation and a specially designed optoacoustic probe for signal detection. The optoacoustic signal induced in the superior sagittal sinus by the nanosecond laser pulses correlated well with actual oxyhemoglobin saturation measured with CO-oximeter. We propose to use a two- or multi- wavelength optoacoustic system for noninvasive continuous monitoring of cerebral venous blood oxygenation. The spectra of effective attenuation coefficient were measured in the range 680-1300 nm for oxy- and deoxygenated whole blood and can be employed for calibration of the system.

Optoacoustic technique for continuous, noninvasive measurement of total hemoglobin concentration: an in vivo study.

We used an optoacoustic technique to measure continuously and noninvasively total hemoglobin concentration in human blood in vivo. An optoacoustic probe, which combined illuminating fibers and a piezoelectric sensitive element, was placed in contact with the skin over the radial artery. Measurements in healthy volunteers agreed well with actual hemoglobin concentration.

The impact of longer-than-average anesthesia times on the billing of academic anesthesiology departments.

UNLABELLED: Academic anesthesiology departments provide clinical services for surgical procedures that have longer-than-average surgical times and correspondingly increased anesthesia times. We examined the financial impact of these longer times in three ways: 1) the estimated loss in revenue if billing were done on a flat-fee system by using industry-averaged anesthesia times; 2) the estimation of incremental operating room (OR) sites necessitated by longer anesthesia times; and 3) the estimated potential gain in billed units if the hours of productivity of current anesthesia time were applied to surgical cases of average duration. Health Care Financing Administration average times per anesthesia procedure code were used as industry averages. Billing data were collected from four academic anesthesiology departments for 1 yr. Each claim billed with ASA units was included except for obstetric anesthesia care. All clinical sites that do not bill with ASA units were excluded. Base units were determined for each anesthesia procedure code. The mean commercial conversion factor (US$45 per ASA unit) for reimbursement was used to estimate the impact in dollar amounts. In all four groups, anesthesia times exceeded the Health Care Financing Administration average. The loss per group in billed ASA units if a flat-fee billing system were used ranged from 18,194 to 31,079 units per group, representing a 5% to 15% decrease (estimated billing decrease of US$818,719 to US$1,398,536 per group). The number of excess OR sites necessitated by longer surgical and anesthesia times ranged from 1.95 to 4.57 OR sites per group. The potential gain in billed units if the hours of productivity of current anesthesia time were applied to surgical cases of average duration was estimated to be from 13,273 to 21,368 ASA units. Longer-than-average anesthesia and surgical times result in extra hours or additional OR sites to be staffed and loss of potential reimbursement for the four academic anesthesiology departments. A flat-fee system would adversely affect academic anesthesiology departments. IMPLICATIONS: We examined the economic impact of longer-than-average anesthesia times on four academic anesthesiology departments in three ways: the estimated loss in revenue under a flat-fee system, the excess operating room sites staffed, and the potential gain in revenue if the surgeries were of average length. These results should be considered both in productivity measurements and strategies for operating room management.

The dynamics of vascular volume and fluid shifts of lactated Ringer's solution and hypertonic-saline-dextran solutions infused in normovolemic sheep.

UNLABELLED: Infusions of hyperosmotic-hyperoncotic solutions such as hypertonic saline dextran (HSD) are used in Europe for resuscitation of traumatic shock and perioperative volume support as an adjunct to conventional isotonic crystalloids. Whereas plasma volume expansion of HSD has been measured at single time points after the intravascular volume expansion, the detailed time course of fluid shifts during and after infusions have not been reported. We compared the time course of volume expansion during and after 30-min infusions of 4 mL/kg HSD and 25 mL/kg lactated Ringer's solution (LR) in normovolemic conscious splenectomized sheep. Peak plasma volume (Evans blue and hemoglobin dilution) expansion was similar for HSD (7.8 +/- 0.9 mL/kg) and the larger sixfold volume of LR (7.2 +/- 0.5 mL/kg). However, 30 min after the 30-min infusion (T60), plasma expansion remained larger after HSD (5.1 +/- 0.9 mL/kg) than after LR (1.7 +/- 0.6 mL/kg). Both solutions caused an equivalent diuresis. Intravascular volume expansion efficiency (VEE), defined as milliliter plasma expansion/milliliter fluid infused at 0 (T30), 30 (T60), and 60 (T90) min after infusion ended was 1.8, 1.3, and 0.8, respectively for HSD, whereas LR provided a VEE of only 0.27, 0.07, and 0.07. The relative expansion efficiency of HSD versus LR, calculated as the ratio (VEE(HSD)/VEE(LR)), was 7-fold that of LR at the end of infusion T30, and 20-fold at T60, but decreased to 9-fold by T120. Intravascular volume dynamic studies of different volume expanders in animals and patients may provide anesthesiologists with a new tool for monitoring the effectiveness of fluid therapy. IMPLICATIONS: Hypertonic saline dextran (HSD) is a new plasma expander recently approved for clinical use in Europe. We compared the plasma volume expansion of HSD versus lactated Ringers (LR) in normovolemic sheep. After a 30 min infusion, HSD was 7 times as effective at expanding volume as an equal volume of LR, but for the next 90 minutes the relative effectiveness of HSD increased to 10-20 times.

Designing meaningful industry metrics for clinical productivity for anesthesiology departments.

IMPLICATIONS: Clinical productivity measurements that account for differences in clinical settings and concurrencies provided more precise comparisons between two anesthesiology groups. The data show that different concurrencies confound the current industry standard, "per full-time equivalent" measurements, whereas "per operating room site" and "per case" measurements allowed for more meaningful comparisons.

Peroxynitrite reduces vasodilatory responses to reduced intravascular pressure, calcitonin gene-related peptide, and cromakalim in isolated middle cerebral arteries.

Vasodilatory responses to progressive reductions in intravascular pressure or to calcitonin gene-related peptide (CGRP) or cromakalim were determined in rodent middle cerebral arteries (MCAs) before and after treatment with peroxynitrite (ONOO-). Middle cerebral artery diameters in isolated, pressurized MCAs were measured as intravascular pressure was reduced from 100 to 20 mm Hg in 20-mm Hg increments before and after inactive ONOO-, pH-adjusted ONOO-, or 10, 20, or 40 micromol/L ONOO- was added to the bath. In other MCAs, responses to CGRP (1 x 10-9 - 5 x 10-8) or cromakalim (3 x 10-8 - 8 x 10-7) were measured before and after the addition of 25 micromol/L ONOO-. Inactive ONOO- (n = 6, P = 0.40), pH-adjusted ONOO- (n = 6, P = 0.29), and 10 micromol/L ONOO- (n = 6, P = 0.88) did not reduce vasodilatory responses to reduced intravascular pressure. Middle cerebral arteries treated with 20 (n = 6, P < 0.0001) and 40 (n = 6, P > 0.0001) micromol/L ONOO- constricted significantly when intravascular pressure was reduced. Vasodilatory responses to CGRP or cromakalim were reduced by ONOO- (P > 0.02, n = 6 and P > 0.01, n = 7, respectively). ONOO- had no effect on vasoconstriction in response to serotonin or vasodilation in response to KCl. These studies demonstrate that ONOO- reduces multiple cerebral vasodilatory responses.

Intraischemic mild hypothermia increases hippocampal CA1 blood flow during forebrain ischemia.

The hippocampal CA1 sector is selectively vulnerable to forebrain ischemia but protected by mild hypothermia. However, the consequence of intraischemic hypothermia on CA1 blood flow during the insult has not been adequately characterized. The effects of mild intraischemic hypothermia on relative changes in regional hippocampal CA1 blood flow were recorded continuously using laser Doppler flowmetry (LDF) during and 30 min after 6 min of forebrain ischemia. Six experimental groups (n=6/group) of fasted male Wistar rats were compared. Groups 1, 3 and 5 consisted of normothermic rats that underwent either 6 (for CBF measurements) and 6 or 10 (for 7 day survival-CA1 neuronal death measurements) min of transient forebrain ischemia using bilateral carotid clamping and hemorrhagic hypotension. Groups 2, 4 and 6 rats were subjected to mild hypothermia (34 degrees C) before, during, and 30 min after 6 (for CBF measurements) and 6 or 10 (for 7 day survival-CA1 neuronal death measurements) min of transient forebrain ischemia. CA1 blood flow and electroencephalogram (EEG) were continuously recorded. During the ischemic insult there were intergroup differences in the magnitude of CBF decreases in the CA1 region. In both groups 1 and 2, CBF returned to preischemic values within 1 min of reperfusion but hypothermic rats had more sustained hyperemia. Hypothermic rats had a quicker recovery of EEG activity and less delayed CA1 neuronal death (group 2 versus 4). These data suggest ischemic blood flow to the CA1 sector was altered by intraischemic mild hypothermia which may contribute to the greater benefit of intraischemic hypothermic neuroprotection.