Blood on board: The development of a prehospital blood transfusion program in a Canadian helicopter emergency medical service.

OBJECTIVE: Prehospital blood transfusion has been adopted by many civilian helicopter emergency medical services agencies, and early outcomes are positive. The Shock Trauma Air Rescue Society operates six bases in Western Canada and started a blood on board process in 2013 in Regina that has expanded to all bases. Two units of O negative packed red blood cells are carried on every mission. We describe the processes and standard work ensuring safe storage, administration, and stewardship of this important resource. METHODS: The packed red blood cells are stored in an inexpensive, reusable temperature controlled cooler at 1°C-6°C. Close collaboration with local transfusion services and adherence to Canadian transfusion standards contributes to safety and sustainability. RESULTS: From October 1, 2013 to October 10, 2017, the Shock Trauma Air Rescue Society administered blood to 431 patients. Of this total, 62.9% received blood carried on our aircraft. A total of 463 blood box units were administered, and the majority of patients (69.0%) received both units. Blood used in Calgary, Alberta was 100% traceable, and only 1.2% of total units dispensed was wasted. The vast majority of unused units were returned to circulation. CONCLUSION: We describe the process to set up and monitor a prehospital blood transfusion program. Our standard work and stewardship processes minimize wastage of blood while keeping it readily available for our critically ill and injured patients.

The mitochondrial division inhibitor Mdivi-1 rescues mammalian neurons from anesthetic-induced cytotoxicity.

BACKGROUND: Concerns have risen regarding the potential side effects of clinical exposure of the pediatric population to inhalational anesthetics, and how they might impact cognitive, learning, and memory functions. However, neither the mechanisms of anesthetic cytotoxicity, nor potential protective strategies, have yet been fully explored. In this study, we examined whether two of the most commonly used inhalational anesthetics, sevoflurane and desflurane, affect neuronal viability and synaptic network assembly between cultured rat cortical neurons. RESULTS: Primary rat cortical neuron cultures were exposed to equipotent sevoflurane or desflurane for 1 hour. Neuron viability, synaptic protein expression, mitochondrial morphology, and neurite growth were assayed with immunostaining and confocal microscopy techniques. The effects of anesthetics on the functional development of neural networks were evaluated with whole-cell patch clamp recordings of spontaneous synaptic currents. Our results demonstrate that an acute exposure to sevoflurane and desflurane inhibits the development of neurite processes, impacts the mitochondria, and compromises synaptic proteins - concomitant with a reduction in synaptic function in mature networks. Interestingly, pretreatment of neurons with a mitochondrial division inhibitor (Mdivi-1) not only protected mitochondria integrity but also played a protective role against anesthetic-induced structural and functional neurotoxicity. CONCLUSIONS: We show that Mdivi-1 likely plays a protective role against certain harmful effects of general anesthetics on primary rat neuronal cultures. In addition, Mdivi-1 alone plays a direct role in enhancing growth and modulating synaptic activity. This study highlights the importance of further study into possible protective agents against anesthetic neurotoxicity.

A novel somatostatin-immunoreactive mossy fiber pathway associated with HSP25-immunoreactive purkinje cell stripes in the mouse cerebellum.

Somatostatin 28 immunoreactivity (Sst28-ir) identifies a specific subset of mossy fiber terminals in the adult mouse cerebellum. By using double-labeling immunohistochemistry, we determined that Sst28-ir is associated with presynaptic mossy fiber terminal rosettes, and not Purkinje cells, Golgi cells, or unipolar brush cells. Sst28-ir mossy fibers are restricted to the central zone (lobules VI/VII) and nodular zone (lobules IX, X) of the vermis, and the paraflocculus and flocculus. Within each transverse zone the mossy fiber terminal fields form a reproducible array of parasagittal stripes. The boundaries of Sst28-ir stripes align with a specific array of Purkinje cell stripes revealed by using immunocytochemistry for the small heat shock protein HSP25. In the cerebellum of the homozygous weaver mouse, in which a subpopulation of HSP25-ir Purkinje cells are located ectopically, the corresponding Sst28-ir mossy fiber projection is also ectopic, suggesting a role for a specific Purkinje cell subset in afferent pattern formation. Likewise, in the scrambler mutant mouse, Sst28-ir mossy fibers show a very close association with HSP25-ir Purkinje cell clusters. HSP25 itself does not appear to be critical for normal patterning, however: in the KJR mouse, which does not express cerebellar HSP25, Sst28 expression appears to be normal. Likewise, the Purkinje cell patterning antigens zebrin II and HSP25 are expressed normally in both Sst- and Sst-receptor knockout mice, suggesting that somatostatinergic transmission is not necessary for Purkinje cell stripe formation.

P2X7-like receptor activation in astrocytes increases chemokine monocyte chemoattractant protein-1 expression via mitogen-activated protein kinase.

Leukocyte infiltration in the CNS after trauma or inflammation is triggered in part by upregulation of the chemokine, monocyte chemoattractant protein-1 (MCP-1), in astrocytes. However the signals that induce the upregulation of MCP-1 in astrocytes are unknown. We have investigated the roles for ATP P2X7 receptor activation because ATP is an intercellular signaling transmitter that is released in both trauma and inflammation and P2X7 receptors are involved in immune system signaling. Astrocytes in primary cell culture and acutely isolated from the hippocampus were immunopositive for P2X7 receptors. In astrocyte cultures, application of the selective P2X7 agonist, benzoyl-benzoyl ATP (Bz-ATP), activated MAP kinases extracellular signal receptor-activated kinase 1 (ERK1), ERK2, and p38. Purinergic antagonists depressed this activation with a profile suggesting P2X7 receptors. Bz-ATP also increased MCP-1 expression in cultured astrocytes, and again P2X7 antagonists prevented this increase. Blocking either the ERK1/ERK2 or the p38 pathway (with PD98059 or SB203580, respectively) significantly inhibited Bz-ATP-induced MCP-1 expression. Coapplication of both antagonists caused a greater depression. We also tested the roles for ATP receptor activation in inducing MCP-1 upregulation in corticectomy, an in vivo model of trauma. This model of cortical trauma was previously shown to increase MCP-1 expression in vivo principally in astrocytes. Suramin, a wide-spectrum purinergic receptor antagonist, significantly depressed the rapid (3 hr) trauma-induced increase in MCP-1 mRNA. These data indicate that purinergic transmitter receptors in astrocytes are important in regulating chemokine synthesis. The regulation of MCP-1 in astrocytes by ATP may be important in mediating communication with hematopoietic inflammatory cells.

Theta-frequency facilitation of AMPA receptor-mediated synaptic currents in the principal cells of the medial septum.

Recent evidence suggests that Ca(2+)-permeable AMPA receptors display rapid, short-lasting current facilitation. In this study, we investigated the properties of AMPA receptor-mediated synaptic currents in medial septal neurons of the rat in an in vitro slice preparation. Immunocytochemistry with a selective antibody to the GluR2 subunit revealed that both choline acetyltransferase-containing and parvalbumin-containing neurons of the medial septum express no detectable GluR2 subunit immunoreactivity. We used whole cell voltage-clamp recordings to measure synaptically evoked AMPA receptor-mediated currents from medial septal neurons following stimulation of midline afferents. The GYKI 52466 (50 microM)- and 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide (NBQX) (20 microM)-sensitive AMPA receptor-mediated component of the synaptic response was isolated by blocking GABA(A)- and N-methyl-D-aspartate receptor-mediated currents with 30 microM bicuculline and 100 microM 2-amino-5-phosphonovaleric acid, respectively. In some cases, patched cells were filled with Lucifer yellow (0.1%) and imaged using 2-photon laser scanning microscopy. AMPA receptor-mediated currents that were observed in large medial septal neurons (20--30 microm) displayed rectification. These currents were sensitive to external application of philanthotoxin-343 (PhTx-343, 50 microM), a potent, high-affinity antagonist of Ca(2+)-permeable, GluR2-lacking AMPA receptors. Rectifying AMPA receptor-mediated currents also displayed a rapid increase in amplitude when evoked five times at low frequency such as 6 Hz. In contrast to currents observed in large medial septal neurons, AMPA-receptor mediated currents evoked in the remaining small (8--11 microm) neurons were nonrectifying and displayed rapid synaptic depression when stimulated five times at 6 Hz. The currents evoked in these cells were unaffected by external application of PhTx-343 and were therefore GluR2-containing AMPA receptors. The results of the present study demonstrate that the principal projection neurons of the medial septum contain PhTx-343-sensitive, GluR2-lacking AMPA receptors that display rapid current facilitation when stimulated at low frequencies.

Hyperthermic induction of the 27-kDa heat shock protein (Hsp27) in neuroglia and neurons of the rat central nervous system.

The 27-kDa heat shock protein (Hsp27) is constitutively expressed in many neurons of the brainstem and spinal cord, is strongly induced in glial cells in response to ischemia, seizures, or spreading depression, and is selectively induced in neurons after axotomy. Here, the expression of Hsp27 was examined in brains of adult rats from 1.5 hours to 6 days after brief hyperthermic stress (core body temperature of 42 degrees C for 15 minutes). Twenty-four hours following hyperthermia, Western blot analysis showed that Hsp27 was elevated in the cerebral cortex, hippocampus, cerebellum, and brainstem. Immunohistochemistry for Hsp27 revealed a time-dependent, but transient, increase in the level of Hsp27 immunoreactivity (Hsp27 IR) in neuroglia and neurons. Hsp27 IR was detected in astrocytes throughout the brain and in Bergmann glia of the cerebellum from 3 hours to 6 days following heat shock. Peak levels were apparent at 24 hours, gradually declining thereafter. In addition, increases in Hsp27 IR were detected in the ependyma and choroid plexus. Hyperthermia induced Hsp27 IR in neurons of the subfornical organ and the area postrema within 3 hours and reached a maximum by 24 hours with a return to control levels 4-6 days after hyperthermia. Specific populations of hypothalamic neurons also showed Hsp27 IR after hyperthermia. These results demonstrate that hyperthermia induces transient expression of Hsp27 in several types of neuroglia and specific populations of neurons. The pattern of induced Hsp27 IR suggests that some of the activated cells are involved in physiological responses related to body fluid homeostasis and temperature regulation.

Cell specific expression of Hsp70 in neurons and glia of the rat hippocampus after hyperthermia and kainic acid-induced seizure activity.

In this study we investigated the time course, cell-type and stress-specific expression of hsp70 mRNA and Hsp70 protein in glial cells and neurons in the rat brain following heat shock treatment and kainic acid-induced status epilepticus. Transcripts for hsp70 were detected in hippocampal homogenates from 1.5 to 6 h following hyperthermia and from 3 to 24 h following kainic acid-induced seizures. In situ hybridization revealed hsp70 mRNA to be region specific and time-dependent following hyperthermia and kainic acid-induced seizures. Western analysis indicated that Hsp70 reached maximal levels at 3 h after hyperthermia and 12 h after kainic acid-induced seizures. Immunohistochemistry revealed low level expression of Hsp70 protein in dentate granule cells at 1.5 and 3 h after hyperthermia. No Hsp70 protein was detected in neurons of the pyramidal cell layer or dentate hilus at any time following hyperthermia. Small Hsp70-immunoreactive cells were detected throughout the hippocampus following hyperthermia that, based on cell size, distribution, and double-labeling with vimentin, were considered to be glia. In contrast, high levels of Hsp70 protein were detected in neurons of the pyramidal cell layer and dentate hilus at 24 h after seizure-inducing kainic acid injection. These results suggest that expression of Hsp70 protein is cell-specific depending on the stressor. In addition, finding high levels of Hsp70 mRNA in the dentate granule cells after hyperthermia, but little or no Hsp70 protein, suggests that the synthesis of the protein is also regulated at the post-transcriptional level following hyperthermia.

Acute administration of cocaine, but not amphetamine, increases the level of synaptotagmin IV mRNA in the dorsal striatum of rat.

Synaptotagmin IV (Syt IV) is an inducible member of a multi-gene family of synaptic vesicle proteins that participate in Ca2+-dependent and Ca2+-independent interactions during membrane trafficking. We have examined the pattern of expression of Syt IV mRNA following the administration of cocaine and amphetamine. A single acute dose of cocaine, but not amphetamine, resulted in a transient increase, as determined by in situ hybridization, in the steady-state level of Syt IV mRNA in the dorsal striatum of rats 1 h after the administration of the drug. No change in the hybridization pattern of the Syt IV-specific probe to other regions of the rat brain were observed following cocaine or amphetamine administration at the time points examined (1, 3, 6, 12 and 24 h). The pattern of synaptotagmin I-(Syt I) specific hybridization remained constant, relative to controls, for both the cocaine- and amphetamine-treated animals. Northern hybridization analysis of mRNA isolated from striatal tissue using oligonucleotide probes specific to Syt I and Syt IV demonstrated that the probes hybridized exclusively to transcripts of the sizes previously reported for these two synaptotagmins and confirmed that the relative level of Syt IV to Syt I mRNA increased following the administration of cocaine but not amphetamine. These results indicate that these drugs have different effects on altering the levels of Syt IV mRNA. This work, in conjunction with earlier work that demonstrated that cocaine and amphetamine have different effects on the expression of immediate early genes such as c-Fos, supports the hypothesis that these psychotropic agents evoke different patterns of gene expression which may lead to alteration in synaptic efficacy.

The effects of hypoxia-ischemia on expression of c-Fos, c-Jun and Hsp70 in the young rat hippocampus.

The expression of c-Fos, c-Jun and Hsp70 was examined in the hippocampus at 6, 12, 24, 48, 72 h, 4, 7 and 42 days following a combination of unilateral common carotid artery ligation and 60 min of systemic hypoxia (8% oxygen, 92% nitrogen) in 25-day-old male rats. While pyknotic cells were not visible in the hippocampus of control animals, pyknosis was evident in the ipsilateral, but not the contralateral hippocampus, of hypoxic-ischemic animals beginning at 24 h post-hypoxia. Immunohistochemical analysis revealed no c-Fos-, c-Jun- or Hsp70-immunoreactivity (IR) in any control animals. However, at 6 h post-hypoxia, Fos- and Jun-IR was evident throughout the injured ipsilateral hippocampus and later appeared throughout the contralateral hippocampus, which never showed signs of pyknosis. In contrast, Hsp70-IR was first observed at 24 h post-hypoxia and was restricted to the injured ipsilateral hippocampus. Hsp70-IR was not, however, limited to dying neurons. H-I/seizure animals did not express these proteins at any time point. These results suggest that, even in irreversibly injured neurons, Fos, Jun and Hsp70 appear to be involved in the aftermath of ischemia but probably do not play a pivotal role in the outcome of H-I compromised cells. Furthermore, compounded injury (H-I/seizure) appears to block the synthesis these proteins.

Differential expression of c-fos, Hsp70 and Hsp27 after photothrombotic injury in the rat brain.

In situ hybridization and immunohistochemistry were used to examine the expression of c-fos, Hsp70 and Hsp27 following photothrombotic injury in the right fronto-parietal cortex of the rat. C-fos mRNA and protein were detected in the entire cerebral cortex on the lesioned side. Hsp70 mRNA accumulation was observed only adjacent and peripheral to the site of the lesion. At 1 h after photothrombotic injury, Hsp70 expression delineates the area of necrosis at 24 h after photothrombotic injury. Hsp27 protein was observed in the ipsilateral cerebral cortex with the exception of the deep layers of the cingulate cortex. In addition, while c-Fos immunoreactivity was localized in cell nuclei, Hsp27 immunoreactivity was detected in the cytoplasm of astrocytes. These results demonstrate that unilateral cortical injury induces changes in gene expression that vary according to cell type and brain region.

A novel seizure-induced synaptotagmin gene identified by differential display.

Systemic administration of kainic acid, a cyclic analogue of glutamate, produces many of the clinical features of human temporal lobe epilepsy and status epilepticus in rats, including the induction of motor convulsions and the degeneration of neurons in the hippocampus and piriform cortex. Differential display PCR was used to identify mRNAs that are differentially expressed between degenerating and nondegenerating tissues in the brain after kainic acid-induced seizure activity. A novel cDNA fragment expressed in the degenerating hippocampus and piriform cortex, but not in the nondegenerating parietal cortex, was identified, cloned, and sequenced. This novel cDNA fragment identified a new member of the synaptotagmin gene family that is rapidly and transiently induced in response to seizure activity. Differential expression of this synaptotagmin gene, syt X, was confirmed by Northern blot analysis and in situ hybridization. This novel, inducible synaptotagmin gene may provide a direct link between seizure-induced neuronal gene expression and subsequent modulation of synaptic structure and function.

Expression of the 27,000 mol. wt heat shock protein following kainic acid-induced status epilepticus in the rat.

Western analysis and immunohistochemistry were used to determine the time-course and the distribution of the 27,000 mol. wt heat shock protein, Hsp27, in rat brain following systemic administration of kainic acid. No Hsp27 immunoreactivity was detected in naive control animals or in rats that failed to develop status epilepticus. Hsp27 immunoreactivity was detected as early as 12 h in the parietal cortex, piriform cortex and the hippocampus of rats that developed status epilepticus. The number of cells expressing Hsp27 and the intensity of Hsp27 immunoreactivity were increased 24 h after kainic acid administration. Hsp27 immunoreactivity was still observed seven days post-kainic acid injection. The morphology of the Hsp27-positive cells and double immunofluorescence against Hsp27 and glial fibrillary acidic protein revealed that Hsp27-positive cells were astrocytes. In addition, the distribution of Hsp27 suggested that astrocytic Hsp27 was dependent on excitation-induced metabolic stress rather than the direct effect of kainic acid on astrocytes.

The inducible 70,000 molecular/weight heat shock protein is expressed in the degenerating dentate hilus and piriform cortex after systemic administration of kainic acid in the rat.

Using both immunohistochemistry and in situ hybridization, we examined the rat brain for the expression of the inducible 70,000 mol. wt heat shock protein, Hsp70, at 3,6,12 and 24 h after systemic administration of kainic acid. In contrast to previous reports, the present study demonstrates that neurons in the regions most susceptible to seizure-induced cell death accumulate both Hsp70 messenger RNA and protein. Neurons in the denate hilus and piriform cortex contained Hsp70 messenger RNA at 6 h and protein at 12 h. These neutrons contained little or no Hsp70 messenger RNA or protein at 24 h when the majority of cells in these area were pyknotic. Injured neurons in areas such as the parietal cortex, which are less susceptible to seizure-induced cell death, expressed and maintained high levels of Hsp70 messenger RNA and protein at 12 and 24 h. This work suggest that Hsp70 messenger RNA and protein are rapidly and transiently expressed in dying neurons, and contradicts the notion that Hsp70 only accumulates in injured neurons that survive.

Antisense Therapeutics in the Central Nervous System : The Induction of c-fos.

Immediate-early genes (IEGs) are members of a class of genes that respond, in many cell types, to a variety of stimuli by rapid, but transient expression (1). Several of these IEGs code for transcription factors and include the widely studied activator protein-1 (AP-1) transcription factor complex believed to be homo- and heterodimeric assemblies of the Fos and Jun families (1-3). IEGs are induced in the central nervous system (CNS) by diverse physiological and pharmacological stimuli, many of which, when presented once or on multiple occasions, can alter the "normal" functioning of the brain in a permanent or semipermanent fashion. Examples of pharmacological stimuli that lead to long-term changes are the highly addictive psychostimulant drugs, amphetamine and cocaine These drugs produce a robust activation of IEGs (e.g., c-fos, jun-B, egr-1) in areas of the brain that are believed to be part of the neural substrates of addiction (4-8) In animal models of epileptogenesis or memory, such as kindling and long-term potentiation (LTP), respectively, electrical stimuli produce activation of IEGs within the brain structures thought to underlie the long-lasting changes associated with these experimental procedures (9-16). IEGs can also be induced by noninvasive stimuli, such as a simple light pulse given to animals in a dark room. The circadian rhythms of animals that are housed in darkened conditions can be shifted by exposing them to a light during then subjective night. Activation of IEGs in such experiments are restricted to the suprachiasmatic nucleus (SCN), which is believed to be the seat of the biological clock (17).

The application of antisense oligonucleotide technology to the brain: some pitfalls.

1. Amphetamine-induced c-fos and egr-1 expression in the striatum was used as a model in which to study the effects of antisense oligodeoxynucleotides (ODNs) directed at c-fos. Using direct infusions of ODNs into the striata of animals we have demonstrated that c-fos antisense ODNs retain most of their biological activity with 2- or 3-base substitutions. The c-fos antisense and mismatch ODNs attenuated Fos immunoreactivity but had little effect on Egr-1 immunoreactivity. 2. In another group of studies examining the role of c-fos in amygdala kindling, we have demonstrated that ODNs cause neurotoxic damage following repeated daily infusions into the amygdala. The damage observed was greatly diminished when the time interval between infusions was extended.

Learning and memory after adrenalectomy-induced hippocampal dentate granule cell degeneration in the rat.

Adrenalectomy (ADX) of normal adult rats causes selective hippocampal dentate granule cell degeneration that is prevented by corticosterone. The ability to destroy this one hippocampal cell type noninvasively made it possible to address the role of the dentate granule cells in learning and memory. Four months after ADX, 31 of 45 rats failed to show obvious granule cell loss and displayed behavior in the Morris water maze that was similar to 16 sham-operated control rats and 16 ADX rats maintained on corticosterone throughout the study. Conversely, 14 of the 45 ADX rats experienced a loss of granule cells that varied from minimal to extensive. Although there were no obvious differences between groups in motoric and motivational characteristics or search strategies, ADX rats with moderate to extensive granule cell loss acquired place learning slightly slower than controls or ADX rats with minimal or no obvious cell loss. Furthermore, the ADX rats with moderate to extensive cell loss were temporarily impaired following alteration of either intramaze or extramaze cues compared to controls. In contrast, the rats with granule cell loss remembered an old place and learned a new place as quickly as controls. These results suggest that a normal complement of dentate granule cell may not be necessary for the acquisition or retention of spatial information in the Morris water maze.

Kindling in the perirhinal cortex.

In vitro experiments have indicated that the perirhinal cortex is highly excitable and its relationship to the basolateral amygdala and piriform cortex is altered by previous amygdala or dorsal hippocampal kindling. As a result, we felt it was important to assess the excitability of the perirhinal cortex in vivo by comparing its kindling profile to that of the basal amygdala, piriform cortex or dorsal hippocampus. We observed that the after-discharge (AD) threshold of the perirhinal cortex was higher than the other 3 structures but the AD duration was not different. Subsequently, the perirhinal cortex kindled more rapidly than the other 3 structures, and with extremely short latencies to onset of forelimb clonus. With the view that synchronized discharge in the perirhinal-piriform area provides the critical trigger for limbic kindled convulsions, the relationship of kindling rate and convulsion latencies and durations between the 4 structures was discussed.

Neonatal 6-OHDA lesions and rearing in complex environments: regional effects on adult brain 14C-2-deoxyglucose uptake revealed by exposure to novel stimulation.

Behavioral and neuromorphological data have suggested at least a partial interaction between the effects of norepinephrine-depleting neonatal 6-OHDA lesions and the effects of rearing in enriched environments. The present study examined the impact of both of these early manipulations upon regional brain uptake of 14C-2-deoxyglucose (14C-2DG) in adulthood. Newborn rats received 6-OHDA (50 mg/kg s.c.) or vehicle and, after weaning at 25 days, were reared in isolated versus enriched conditions. Regional brain 14C-2DG uptake was then examined at 70-80 days of age--either in the home cage or while animals were being exposed to novel, presumably arousing, stimulation. Ninety-seven brain regions were examined in eight separate groups. Results indicated that (1) Under baseline conditions, neither neonatal 6-OHDA nor differential rearing conditions produced widespread alterations in regional brain 14C-2DG uptake profiles. An overall enrichment effect was seen on only five brain areas, with rats reared in enriched environments showing lower levels of 14C-2DG uptake (-20% to -30%) than isolated rats. Neonatal 6-OHDA produced no main effect on 14C-2DG uptake in any brain region. (2) In contrast, when 14C-2DG uptake was assessed during exposure to a novel environment, five brain areas showed differential 14C-2DG uptake in 6-OHDA-treated rats, and 20 brain areas showed differential uptake in rats reared in enriched conditions. (3) No significant interaction effect on brain regional 14C-2DG uptake was observed between neonatal 6-OHDA and environmental complexity factors. These results are consistent with the notion that enduring effects of rearing and early 6-OHDA treatment may, independently, relate to a general reactivity factor. They also indicate that some effects of early neurochemical injury and subsequent experiential factors may not be apparent under normal resting conditions, but only become evident in the presence of appropriate "activating" stimulation.