Storage with ethanol attenuates the red blood cell storage lesion.

BACKGROUND: Current management of hemorrhagic shock relies on control of surgical bleeding along with resuscitation with packed red blood cells and plasma in a 1-to-1 ratio. Transfusion, however, is not without consequence as red blood cells develop a series of biochemical and physical changes during storage termed "the red blood cell storage lesion." Previous data has suggested that ethanol may stabilize the red blood cell membrane, resulting in improved deformability. We hypothesized that storage of packed red blood cells with ethanol would alter the red blood cell storage lesion. METHODS: Mice underwent donation and storage of red blood cells with standard storage conditions in AS-3 alone or ethanol at concentrations of 0.07%, 0.14%, and 0.28%. The red blood cell storage lesion parameters of microvesicles, Band-3, free hemoglobin, annexin V, and erythrocyte osmotic fragility were measured and compared. In additional experiments, the mice underwent hemorrhage and resuscitation with stored packed red blood cells to further evaluate the in vivo inflammatory impact. RESULTS: Red blood cells stored with ethanol demonstrated decreased microvesicle accumulation and Band-3 levels. There were no differences in phosphatidylserine or cell-free hemoglobin levels. After hemorrhage and resuscitation with packed red blood cells stored with 0.07% ethanol, mice demonstrated decreased serum levels of interleukin-6, macrophage inflammatory protein-1α, keratinocyte chemokine, and tumor necrosis factor α compared to those mice receiving packed red blood cells stored with additive solution-3. CONCLUSION: Storage of murine red blood cells with low-dose ethanol results in decreased red blood cell storage lesion severity. Resuscitation with packed red blood cells stored with 0.07% ethanol also resulted in a decreased systemic inflammatory response in a murine model of hemorrhage.

Improving packed red blood cell storage with a high-viscosity buffered storage solution.

BACKGROUND: Massive transfusion with older packed red blood cells is associated with increased morbidity and mortality. As packed red blood cells age, they undergo biochemical and structural changes known as the storage lesion. We developed a novel solution to increase viscosity in stored packed red blood cells. We hypothesized that packed red blood cell storage in this solution would blunt storage lesion formation and mitigate the inflammatory response after resuscitation. METHODS: Blood was obtained from 8- to 10-week-old C57BL/6 male donor mice or human volunteers and stored as packed red blood cell units for 14 days for mice or 42 days for humans in either standard AS-3 storage solution or EAS-1587, the novel packed red blood cell storage solution. Packed red blood cells were analyzed for microvesicles, cell-free hemoglobin, phosphatidylserine, band-3 protein, glucose utilization, and osmotic fragility. Additional mice underwent hemorrhage and resuscitation with packed red blood cells stored in either AS-3 or EAS-1587. Serum was analyzed for inflammatory markers. RESULTS: Murine packed red blood cells stored in EAS-1587 demonstrated reductions in microvesicle and cell-free hemoglobin accumulation as well as preserved band-3 expression, increase glucose utilization, reductions in phosphatidylserine expression, and susceptibility to osmotic stress. Serum from mice resuscitated with packed red blood cells stored in EAS-1587 demonstrated reduced proinflammatory cytokines. Human packed red blood cells demonstrated a reduction in microvesicle and cell-free hemoglobin as well as an increase in glucose utilization. CONCLUSION: Storage of packed red blood cells in a novel storage solution mitigated many aspects of the red blood cell storage lesion as well as the inflammatory response to resuscitation after hemorrhage. This modified storage solution may lead to improvement of packed red blood cell storage and reduce harm after massive transfusion.

Effect of dexmedetomidine on sevoflurane-induced neurodegeneration in neonatal rats.

BACKGROUND: Structural brain abnormalities in newborn animals after prolonged exposure to all routinely used general anaesthetics have raised substantial concerns for similar effects occurring in millions of children undergoing surgeries annually. Combining a general anaesthetic with non-injurious sedatives may provide a safer anaesthetic technique. We tested dexmedetomidine as a mitigating therapy in a sevoflurane dose-sparing approach. METHODS: Neonatal rats were randomised to 6 h of sevoflurane 2.5%, sevoflurane 1% with or without three injections of dexmedetomidine every 2 h (resulting in 2.5, 5, 10, 25, 37.5, or 50 µg kg-1 h-1), or fasting in room air. Heart rate, oxygen saturation, level of hypnosis, and response to pain were measured during exposure. Neuronal cell death was quantified histologically after exposure. RESULTS: Sevoflurane at 2.5% was more injurious than at 1% in the hippocampal cornu ammonis (CA)1 and CA2/3 subfields; ventral posterior and lateral dorsal thalamic nuclei; prefrontal, retrosplenial, and somatosensory cortices; and subiculum. Although sevoflurane 1% did not provide complete anaesthesia, supplementation with dexmedetomidine dose dependently increased depth of anaesthesia and diminished responses to pain. The combination of sevoflurane 1% and dexmedetomidine did not reliably reduce neuronal apoptosis relative to an equianaesthetic dose of sevoflurane 2.5%. CONCLUSIONS: A sub-anaesthetic dose of sevoflurane combined with dexmedetomidine achieved a level of anaesthesia comparable with that of sevoflurane 2.5%. Similar levels of anaesthesia caused comparable programmed cell death in several developing brain regions. Depth of anaesthesia may be an important factor when comparing the neurotoxic effects of different anaesthetic regimens.

Washing packed red blood cells decreases red blood cell storage lesion formation.

BACKGROUND: Transfusion of blood products is the ideal resuscitative strategy after hemorrhage. Unfortunately, older packed red blood cells have been associated with increased morbidity and mortality after massive transfusion. These packed red blood cells accumulate biochemical and structural changes known as the red blood cell storage lesions. The effect of washing on the formation of red blood cell storage lesions is unknown. We hypothesized that washing packed red blood cells during storage would decrease the development of the red blood cell storage lesions. METHODS: Blood from 8- to 10-week-old male mice donors was stored as packed red blood cells for 14 days. A subset of packed red blood cells were washed with phosphate-buffered saline on storage day 7 and resuspended in AS-1 solution for an additional 7 days as washed packed red blood cells. Subsequently, the packed red blood cells were analyzed for microvesicle release, band-3 erythrocyte membrane integrity protein (Band-3), expression of phosphatidylserine, cell viability (calcein), accumulation of cell-free hemoglobin, and osmotic fragility. RESULTS: In the washed packed red blood cells group, there was less microvesicle accumulation, greater Band-3 expression, less phosphatidylserine expression, a decrease in cell-free hemoglobin accumulation, and a decrease in osmotic fragility, but no differences in red blood cells viability. CONCLUSION: Washing packed red blood cells during storage decreases the accumulation of red blood cell storage lesions. This strategy may lessen the sequelae associated with transfusion of older packed red blood cells.

Expired But Not Yet Dead: Examining the Red Blood Cell Storage Lesion in Extended-Storage Whole Blood.

ABSTRACT: Whole blood is a powerful resuscitation strategy for trauma patients but has a shorter shelf life than other blood products. The red blood cell storage lesion in whole blood has not previously been investigated beyond the standard storage period. In the present study, we hypothesized that erythrocytes in stored whole blood exhibit similar aspects of the red blood cell storage lesion and that transfusion of extended storage whole blood would not result in a more severe inflammatory response after hemorrhage in a murine model. To test this hypothesis, we stored low-titer, O-positive, whole blood units, and packed red blood cells (pRBCs) for up to 42 days, then determined aspects of the red blood cell storage lesion. Compared with standard storage pRBCs, whole blood demonstrated decreased microvesicle and free hemoglobin at 21 days of storage and no differences in osmotic fragility. At 42 days of storage, rotational thromboelastometry demonstrated that clotting time was decreased, alpha angle was increased, and clot formation time and maximum clot firmness similar in whole blood as compared with pRBCs with the addition of fresh frozen plasma. In a murine model, extended storage whole blood demonstrated decreased microvesicle formation, phosphatidylserine, and cell-free hemoglobin. After hemorrhage and resuscitation, TNF-a, IL-6, and IL-10 were decreased in mice resuscitated with whole blood. Red blood cell survival was similar at 24 h after transfusion. Taken together, these data suggest that red blood cells within whole blood stored for an extended period of time demonstrate similar or reduced accumulation of the red blood cell storage lesion as compared with pRBCs. Further examination of extended-storage whole blood is warranted.

Innate coagulability changes with age in stored packed red blood cells.

BACKGROUND: Packed red blood cell (pRBC) units administered during resuscitation from hemorrhagic shock are of varied storage ages. We have previously shown that RBC-derived microparticles' impact on thrombogenesis. However, the impact of storage age on pRBC coagulability is unknown. Therefore, we sought to investigate the effect of storage age on innate coagulability and aggregability of stored pRBCs. METHODS: pRBCs prepared from male C57BL/6J mice were stored in Additive Solution-3 according to our standardized murine blood banking protocols for 14 days. Rotational thromboelastometry (ROTEM) was used to assess the innate coagulation status of fresh and 14-day old pRBCs. Viscoelastic coagulation parameters of clotting time (CT), clot formation time (CFT), alpha angle, and maximum clot firmness (MCF) were analyzed to determine coagulability. Plasma was added to the fresh pRBCs and 15-day old pRBCs to determine if the storage-associated coagulopathy was reversible with plasma. Statistical analyses were conducted with a Student's t-test. RESULTS: Fifteen-day old pRBCs demonstrated a significant reduction in MCF (10.3 vs. 24.4 mm, P-value <0.001) and alpha angle (6.0 vs. 27.2 degrees, P-value <0.001) as well as significant prolongation of CFT and CT (1126.5 vs. 571.4 s, P-value <0.001) compared to fresh pRBCs. FFP addition to 15-day old and fresh pRBCs, demonstrated a significant reduction in MCF and persistent prolongation of CFT. This suggests that pRBCs lost coagulability as they aged and this deficit was not completely corrected by plasma administration. CONCLUSIONS: Storage duration may be an important factor in coagulation potential of pRBCs. Transfusion with older pRBCs may contribute to coagulopathy in massively transfused patients.

Save it-don't waste it! Maximizing utilization of erythrocytes from previously stored whole blood.

BACKGROUND: Recent military and civilian experience suggests that fresh whole blood may be the preferred for treatment of hemorrhagic shock, but its use is limited by its 21-day shelf life. The red blood cell storage lesion and coagulation status of packed red blood cells (pRBCs) salvaged from expired whole blood are unknown. We hypothesized that pRBCs can be salvaged from previously stored whole blood. METHODS: Cold stored, low-titer, O-positive, nonleukoreduced, whole blood units were obtained at 21 days of storage. Erythrocytes were separated by centrifugation, resuspended in AS-3, and stored for 21 additional days as salvaged pRBCs. The red blood cell storage lesion parameters of microvesicles, Band-3, free hemoglobin, annexin V, and erythrocyte osmotic fragility were measured and compared with pRBCs prepared at the time of donation and stored in AS-3 for 42 days (standard pRBCs). In additional experiments, murine pRBCs were prepared from expired whole blood units and compared with those stored under standard conditions. Mice underwent hemorrhage and resuscitation with standard and salvaged pRBC units, and serum cytokines and free hemoglobin were determined. RESULTS: There were no significant differences in microvesicle formation or cell-free hemoglobin concentration between salvaged and standard pRBCs. There was decreased Band-3 and increased phosphatidylserine in the salvaged units as well as greater osmotic fragility. Salvaged pRBCs maintained consistent clot firmness. After hemorrhage and resuscitation in a murine model, salvaged pRBCs did not demonstrate increased serum cytokine levels. CONCLUSION: Salvaged pRBCs from previously stored whole blood accumulate the red blood cell storage lesion in a similar fashion to standard pRBCs and maintain consistent coagulability when reconstituted with plasma. Salvaged pRBCs are not associated with an increased inflammatory response when used for resuscitation in a murine model. Salvaged pRBCs may be a viable product for utilization in the treatment of traumatic hemorrhagic shock.

Developmental stage-dependent impact of midazolam on calbindin, calretinin and parvalbumin expression in the immature rat medial prefrontal cortex during the brain growth spurt.

BACKGROUND: Human epidemiological data suggest a link between anesthesia exposure in early postnatal life and subsequent lasting neurocognitive alterations. Understanding the underlying mechanisms of this potential association is of paramount importance in an attempt to develop protective strategies. While general anesthetics are powerful modulators of GABAergic neurotransmission, little is known about the impact of these drugs on developing GABAergic networks. Here we addressed this issue by evaluating the impact of a 6-h-long midazolam exposure on the development of calbindin-, calretinin- and parvalbumin-expressing GABAergic interneurons. METHODS: Physiological expression patterns of calbindin-, calretinin-, and parvalbumin-positive neurons as well as the impact of a 6-h-long midazolam exposure on these cell populations were evaluated in the medial prefrontal cortex of Wistar rats at defined stages of the brain growth spurt using stereological analysis. Activated caspase-3 immunohistochemistry was used to quantify apoptotic death in controls and midazolam-treated subjects. RESULTS: In control animals, the number of parvalbumin expressing cells significantly (p<0.01) increased while those of calbindin positive populations significantly (p<0.01) decreased between postnatal day 10 and 20. Expression of calretinin remained constant during this period. Immediately following exposure, midazolam induced neuroapoptosis at both early (postnatal day 5, p=0.016) and later (postnatal day 15, p=0.025) stages of brain development. While this did not diminish overall neuronal density in the medial prefrontal cortex, exposure at P5 led to a subsequent increase in the number of parvalbumin positive neurons in lower cortical layers, and midazolam administration at P15 increased the number of both parvalbumin and calretinin expressing neurons 5 days following exposure. CONCLUSION: These observations demonstrate that midazolam exposure can impair the physiological differentiation patterns of GABAergic interneurons during the brain growth spurt. Considering the important role of GABAergic networks in neuronal physiology, these data provide us with one potential mechanism that could account for the lasting neurobehavioral and cognitive deficits observed in the context of anesthesia exposure in the early postnatal period.

Cell age-specific vulnerability of neurons to anesthetic toxicity.

OBJECTIVE: Anesthetics have been linked to widespread neuronal cell death in neonatal animals. Epidemiological human studies have associated early childhood anesthesia with long-term neurobehavioral abnormalities, raising substantial concerns that anesthetics may cause similar cell death in young children. However, key aspects of the phenomenon remain unclear, such as why certain neurons die, whereas immediately adjacent neurons are seemingly unaffected, and why the immature brain is exquisitely vulnerable, whereas the mature brain seems resistant. Elucidating these questions is critical for assessing the phenomenon's applicability to humans, defining the susceptible age, predicting vulnerable neuronal populations, and devising mitigating strategies. METHODS: This study examines the effects of anesthetic exposure on late- and adult-generated neurons in newborn, juvenile, and adult mice, and characterizes vulnerable cells using birth-dating and immunohistochemical techniques. RESULTS: We identify a critical period of cellular developmental during which neurons are susceptible to anesthesia-induced apoptosis. Importantly, we demonstrate that anesthetic neurotoxicity can extend into adulthood in brain regions with ongoing neurogenesis, such as dentate gyrus and olfactory bulb. INTERPRETATION: Our findings suggest that anesthetic vulnerability reflects the age of the neuron, not the age of the organism, and therefore may potentially not only be relevant to children but also to adults undergoing anesthesia. This observation further predicts differential heightened regional vulnerability to anesthetic neuroapoptosis to closely follow the distinct regional peaks in neurogenesis. This knowledge may help guide neurocognitive testing of specific neurological domains in humans following exposure to anesthesia, dependent on the individual's age during exposure.

Desflurane, isoflurane, and sevoflurane provide limited neuroprotection against neonatal hypoxia-ischemia in a delayed preconditioning paradigm.

BACKGROUND: The volatile anesthetics desflurane, isoflurane, and sevoflurane have been found to produce neuroprotection in various paradigms. The authors used these agents in a delayed preconditioning model to test the hypothesis that they could provide neuroprotection against neonatal hypoxia-ischemia as assessed by a battery of behavioral tests. METHODS: Institutional Animal Care and Use Committee approval was obtained. A total of 140, C57-129T2 F1 hybrid 9-day-old mice were randomized to 3 h of preconditioning with room air (Group Sham and Group HI), 8.4% desflurane in 40% oxygen (Group D), 1.8% isoflurane (Group I), or 3.1% sevoflurane (Group S). Twenty-four hours later, the Group HI, D, I, and S mice had 60 min of hypoxia-ischemia, and Group Sham had 60 min of sham HI. Surviving animals had behavioral testing, including open field activity, acoustic startle, prepulse inhibition, rotorod, novel object recognition, water mazes, and apomorphine challenge. Histologic analysis was also performed. RESULTS: Mice in Groups D, I, and S performed better than Group HI and similarly to Group Sham on novel object recognition and apomorphine challenge and better than Group HI but not as well as Group Sham on cued maze testing. All mice exposed to hypoxia-ischemia performed worse than Group Sham on the spatially oriented water mazes with no difference among groups. Histologic sections did not show any significant effect of preconditioning on injury scores. CONCLUSIONS: Volatile agent preconditioning partially protects perirhinal cortex and striatal dependent functions against moderate to severe neonatal hypoxia-ischemia.

Metallothionein I,II deficient mice do not exhibit significantly worse long-term behavioral outcomes following neonatal hypoxia-ischemia: MT-I,II deficient mice have inherent behavioral impairments.

Metallothionein I and II are small metal binding proteins with a high affinity for zinc. They are found in the CNS and are thought to play a role in modulating the effects of free zinc. We hypothesized that MT-I,II deficient mice would have more neurological deficits both functionally and anatomically following a neonatal hypoxic-ischemic (HI) insult than wild-type mice subjected to the same insult. Forty wild-type and 40 MT-I,II deficient C57 X 129T2 F1 P10 mice were randomized to either 45 min of HI or sham HI. Beginning on P50, the mice were given a series of behavioral tests including locomotor activity, novel object recognition, Morris water maze (cued, hidden platform, reduced platform), a 2-week-delayed probe trial and an apomorphine-induced rotation test. At the conclusion of testing, the brains were removed for histological analysis including staining with NeuN and GFAP to assess neuronal loss and reactive gliosis. There were no significant differences in functional or anatomic measures between the wild-type HI mice and the MT-I,II deficient HI mice. The MT-I,II deficient mice exhibited an impaired rate of learning in the spatially oriented mazes but once learned retained the information as well as the wild-type mice. The absence of functional MT-I,II proteins does not result in significantly worse injury following 45 min of HI on P10. The MT-I,II deficient mice have baseline impairments in spatial learning but not retention.

Isoflurane-delayed preconditioning reduces immediate mortality and improves striatal function in adult mice after neonatal hypoxia-ischemia.

BACKGROUND: Exposure to hypoxia and isoflurane (Iso) before hypoxia-ischemia has been found to be neuroprotective in neonatal rats. We investigated the long-term effects of delayed preconditioning with Iso, hypoxia, or room air on motor and cognitive function in mice that had 65 min of hypoxia-ischemia on postnatal day 10. METHODS: Nine-day-old C57x129T2 F1 mice received either 1.8% Iso, hypoxic (10% O2 in N2), or sham (room air) preconditioning. The following day, the mice were subjected to permanent right common carotid ligation or sham ligation followed by 65 min of hypoxia, or room air. At 70 days of age, learning was tested using a series of Morris water maze tests. Striatal function was assessed by response to apomorphine injection. Histological analysis was performed on adult brain (P120) sections of striatum and dorsal hippocampus. RESULTS: Iso preconditioning 24 h before severe neonatal hypoxia-ischemia reduced preweaning mortality from 20% to 0% (P < 0.04) and improved striatal function in adult mice, as assessed by circling after apomorphine injection (P < 0.028), but no improvements in performance were noted in the spatial-reference memory water maze tests. Hypoxic preconditioning improved learning relative to the sham-preconditioned group on the hidden maze, but not the more difficult reduced maze test of spatial memory. It had no significant effect on preweaning mortality and apomorphine response. Histologic analysis showed the hippocampus of non-preconditioned and Iso-preconditioned animals to be equally injured. CONCLUSION: Iso and hypoxia confer selective functional neuroprotection in a delayed preconditioning paradigm in neonatal mice.

Relationship between myelin production and dopamine synthesis in the PKU mouse brain.

Phenylketonuria is caused by specific mutations in the phenylalanine hydroxylase gene and is characterized by elevated blood phenylalanine levels, hypomyelination in forebrain structures, reduced dopamine levels, and cognitive difficulties. To determine whether brain tyrosine levels and/or myelination play a role in the up-regulation of dopamine, phenylketonuric mice were placed on a low phenylalanine diet for 4 weeks and as blood phenylalanine levels dropped to normal, the relationships between phenylalanine, tyrosine, dopamine, myelin proteins, and axonal proteins in frontal cortex and striatum were determined using gas chromatography mass spectrometry, histology, and western blotting techniques. Blood phenylalanine rapidly decreased from an eight-fold elevation to near control levels, and blood tyrosine gradually rose from about 50% to near normal values. In frontal cortex and striatum, phenylalanine levels dropped to 2- and 1.5-fold elevations above control, respectively, and tyrosine levels increased but remained less than 70% of control in both structures. In frontal cortex, increases in dopamine and myelin basic protein occurred in a similar biphasic pattern, reaching near normal levels by week 4. In striatum, dopamine and MBP dramatically increased to near normal levels in the first week. Myelination was confirmed histologically and by western blot quantification of phosphorylated neurofilaments. In summary, our results showed: (i) an increase in dopamine despite low brain tyrosine levels and (ii) similar recovery patterns for myelination and dopamine. Since myelin/axonal interactions trigger signaling pathways that result in axonal maturation, we speculate that this interaction also may trigger signals that up-regulate neurotransmitter synthesis.