Cellular and Biochemical Effects of Combined X-Ray Radiation and Storage on Whole Blood.

BACKGROUND: Evaluating the impact of ionizing radiation on stored blood is relevant since blood banks are major assets in emergency conditions such as radiation incident/attack. This study aimed to fill our knowledge gap of combined radiation and storage effects on blood. METHODS: Blood collected from 16 anesthetized rats was anticoagulated, aliquoted into storage bags, and assigned to 8 groups using protocols combining storage (1-day vs 3-day 4oC) plus irradiation (75 Gy vs 0 Gy - control). Bags were positioned inside an X-ray irradiator (MultiRad-350). Complete blood count, differential white blood cell count, biochemistry, and hemostasis were analyzed (≥7 bags/group). RESULTS: Na+, bicarbonate, glucose, and pH significantly reduced, while K+, Cl-, and lactate increased by storage. Coagulation measures were not significantly altered after radiation. White blood cell count and most cell types were numerically reduced after radiation, but changes were statistically significant only for monocytes. No significant alterations were noted in aggregation or rotational thromboelastometry parameters between irradiated and control. CONCLUSIONS: Evaluating cellular/biochemical parameters aids in assessing stored blood adequacy after radiation. Data suggest that fresh or cold-stored blood can sustain up to 75 Gy without major critical parameter changes and may remain suitable for use in critically ill patients in military/civilian settings.

Cremaster muscle perfusion, oxygenation, and heterogeneity revealed by a new automated acquisition system in a rodent model of prolonged hemorrhagic shock.

Local blood flow/oxygen partial pressure (Po2) distributions and flow-Po2 relationships are physiologically relevant. They affect the pathophysiology and treatment of conditions like hemorrhagic shock (HS), but direct noninvasive measures of flow, Po2, and their heterogeneity during prolonged HS are infrequently presented. To fill this void, we report the first quantitative evaluation of flow-Po2 relationships and heterogeneities in normovolemia and during several hours of HS using noninvasive, unbiased, automated acquisition. Anesthetized rats were subjected to tracheostomy, arterial/venous catheterizations, cremaster muscle exteriorization, hemorrhage (40% total blood volume), and laparotomy. Control animals equally instrumented were not subjected to hemorrhage/laparotomy. Every 0.5 h for 4.5 h, noninvasive laser speckle contrast imaging and phosphorescence quenching were employed for nearly 7,000 flow/Po2 measurements in muscles from eight animals, using an automated system. Precise alignment of 16 muscle areas allowed overlapping between flow and oxygenation measurements to evaluate spatial heterogeneity, and repeated measurements were used to estimate temporal heterogeneity. Systemic physiological parameters and blood chemistry were simultaneously assessed by blood samplings replaced with crystalloids. Hemodilution was associated with local hypoxia, but increased flow prevented major oxygen delivery decline. Adding laparotomy and prolonged HS resulted in hypoxia, ischemia, decreased tissue oxygen delivery, and logarithmic flow/Po2 relationships in most regions. Flow and Po2 spatial heterogeneities were higher than their respective temporal heterogeneities, although this did not change significantly over the studied period. This quantitative framework establishes a basis for evaluating therapies aimed at restoring muscle homeostasis, positively impacting outcomes of civilian and military trauma/HS victims.NEW & NOTEWORTHY This is the first study on flow-Po2 relationships during normovolemia, hemodilution, and prolonged hemorrhagic shock using noninvasive methods in multiple skeletal muscle areas of monitored animals. Automated flow/Po2 measurements revealed temporal/spatial heterogeneities, hypoxia, ischemia, and decreased tissue oxygen delivery after trauma/severe hemorrhage. Hemodilution was associated with local hypoxia, but hyperemia prevented a major decline in oxygen delivery. This framework provides a quantitative basis for testing therapeutics that positively impacts muscle homeostasis and outcomes of trauma/hemorrhagic shock victims.

Automated noninvasive evaluation of blood flow and oxygenation in rats integrated with systemic physiological monitoring.

BACKGROUND: Many studies evaluating blood flow and oxygen partial pressure (PO2) do not directly measure both parameters, are confined to few locations/microvessels, and depend on investigator's selection of measuring sites. Moreover, clinically/physiologically relevant systemic parameters are not simultaneously recorded. We implemented an automated system for prolonged blood flow/PO2 acquisition in large areas while collecting relevant systemic information. METHODS: In anesthetized animals, cardiorespiratory parameters were continuously recorded. Other data were collected at baseline and hourly after 4 hours of hemorrhagic shock. A cremaster muscle was spread over a pedestal fixed to a motorized stage. One 2-dimensional tissue scan allowed 16 noninvasive PO2 measurements using oxygen-dependent phosphorescence quenching and fiber optics. Blood flow was estimated using laser speckle contrast imaging in the same areas used for PO2 measurements. At each timepoint, blood was sampled for extensive biochemistry/coagulation profile. RESULTS: The system was used successfully by different operators. A set of flow/PO2 measurements was completed in less than 90 seconds. Muscle flow and PO2 correlated with some but not several systemic parameters, emphasizing the importance of performing both local and systemic evaluations. CONCLUSION: System advantages include integration between local and over 40 systemic parameters, unbiased data collection/analysis, improved performance/sampled area, easy expansion, implementation and maintenance, no customized programming, and simplified training. Combining this system with trauma/prolonged HS models will enhance our ability to investigate tissue stability and select better resuscitation strategies to improve outcomes and survival. LEVEL OF EVIDENCE: Diagnostic test, level V.

Predicting the responses of repetitively firing neurons to current noise.

We used phase resetting methods to predict firing patterns of rat subthalamic nucleus (STN) neurons when their rhythmic firing was densely perturbed by noise. We applied sequences of contiguous brief (0.5-2 ms) current pulses with amplitudes drawn from a Gaussian distribution (10-100 pA standard deviation) to autonomously firing STN neurons in slices. Current noise sequences increased the variability of spike times with little or no effect on the average firing rate. We measured the infinitesimal phase resetting curve (PRC) for each neuron using a noise-based method. A phase model consisting of only a firing rate and PRC was very accurate at predicting spike timing, accounting for more than 80% of spike time variance and reliably reproducing the spike-to-spike pattern of irregular firing. An approximation for the evolution of phase was used to predict the effect of firing rate and noise parameters on spike timing variability. It quantitatively predicted changes in variability of interspike intervals with variation in noise amplitude, pulse duration and firing rate over the normal range of STN spontaneous rates. When constant current was used to drive the cells to higher rates, the PRC was altered in size and shape and accurate predictions of the effects of noise relied on incorporating these changes into the prediction. Application of rate-neutral changes in conductance showed that changes in PRC shape arise from conductance changes known to accompany rate increases in STN neurons, rather than the rate increases themselves. Our results show that firing patterns of densely perturbed oscillators cannot readily be distinguished from those of neurons randomly excited to fire from the rest state. The spike timing of repetitively firing neurons may be quantitatively predicted from the input and their PRCs, even when they are so densely perturbed that they no longer fire rhythmically.

Firing rate and pattern heterogeneity in the globus pallidus arise from a single neuronal population.

Intrinsic heterogeneity in networks of interconnected cells has profound effects on synchrony and spike-time reliability of network responses. Projection neurons of the globus pallidus (GPe) are interconnected by GABAergic inhibitory synapses and in vivo fire continuously but display significant rate and firing pattern heterogeneity. Despite being deprived of most of their synaptic inputs, GPe neurons in slices also fire continuously and vary greatly in their firing rate (1-70 spikes/s) and in regularity of their firing. We asked if this rate and pattern heterogeneity arises from separate cell types differing in rate, local synaptic interconnections, or variability of intrinsic properties. We recorded the resting discharge of GPe neurons using extracellular methods both in vivo and in vitro. Spike-to-spike variability (jitter) was measured as the standard deviation of interspike intervals. Firing rate and jitter covaried continuously, with slow firing being associated with higher variability than faster firing, as would be expected from heterogeneity arising from a single physiologically distinct cell type. The relationship between rate and jitter was unaffected by blockade of GABA and glutamate receptors. When the firing rate of individual neurons was altered with constant current, jitter changed to maintain the rate-jitter relationship seen across neurons. Long duration (30-60 min) recordings showed slow and spontaneous bidirectional drift in rate similar to the across-cell heterogeneity. Paired recordings in vivo and in vitro showed that individual cells wandered in rate independently of each other. Input conductance and rate wandered together, in a manner suggestive that both were due to fluctuations of an inward current.

Slow spike frequency adaptation in neurons of the rat subthalamic nucleus.

Neurons of the subthalamic nucleus (STN) are very sensitive to applied currents, firing at 10-20/s during spontaneous activity, but increasing to peak firing rates of 200/s with applied currents <0.5 nA. They receive a powerful tonic excitatory input from neurons in the cerebral cortex, yet in vivo maintain an irregular firing rate only slightly higher than the autonomous firing rate seen in slices. Spike frequency adaptation acts to normalize background firing rate by removing slow trends in firing due to changes in average input. Subthalamic neurons have been previously described as showing little spike frequency adaptation, but this was based on tests using brief stimuli. We applied long-duration depolarizing current steps to STN neurons in slices and observed a very strong spike frequency adaptation with a time constant of 20 s and that recovered at a similar rate. This adaptation could return firing to near-baseline levels during prolonged current pulses that transiently drove the cells at high rates. The current responsible for adaptation was studied in voltage clamp during and after high-frequency driving of the cell and was determined to be a slowly accumulating K(+) current. This current was independent of calcium or sodium entry and could be induced with long-duration voltage steps after blockade of action potentials. In addition to the adaptation current, driven firing produced slow inactivation of the persistent Na(+) current, which also contributed to the reduced excitability of STN cells during and after driven firing.