Effect of Hyperoxia on Resuscitation of Experimental Combined Traumatic Brain Injury and Hemorrhagic Shock in Mice

NEUROLOGIC deficits following traumatic brain injury (TBI) are caused by the primary mechanical injury and subsequent cascade of biochemical, metabolic, and cellular derangements encompassing the secondary injury. In civilian and military settings, TBI is often seen not in isolation but in combination with other insults (polytrauma) that exaggerate the secondary injury.1,2The occurrence of hypotension is associated with doubled mortality and worsened morbidity after TBI.3Coupled with reduced cerebral blood flow acutely following TBI,4–6hemorrhagic hypotension likely exacerbates cerebral hypoxia.7,8Sustained brain tissue oxygen tension (PbtO2) levels less than 15 mmHg or a PbtO2less than 6 mmHg for any period of time following TBI is associated with increased mortality.9Administration of supplemental oxygen after TBI in an attempt to improve oxygenation of vulnerable brain tissue in various settings has been used clinically with mixed results.10–13In the setting of TBI with superimposed hemorrhagic shock (HS), however, it is unknown whether administration of supplemental oxygen improves resuscitation and outcomes.Varying the fraction of inspired oxygen after different types of brain injury is currently under intense debate.14Hyperoxic resuscitation of a focal cerebral injury has been shown to be neuroprotective in experimental studies by improving penumbral blood flow and maintaining penumbral oxygenation, decreasing infarct size, and ultimately improving outcomes.15–20In clinical stroke studies, early application of normobaric hyperoxia increased magnetic resonance imaging evidence of aerobic metabolism and neuronal stability as well as transient clinical improvement.21,22At the other end of the spectrum, however, the ischemia–reperfusion injury that occurs from administration of 100% oxygen on return of spontaneous circulation after cardiac arrest could be detrimental, particularly with regard to neurologic outcomes.23Kilgannon et al. recently showed arterial hyperoxia to be an independent predictor of in-hospital mortality after resuscitation from cardiac arrest.24The surge of reactive oxygen species upon hyperoxic reperfusion leads to neuronal death from damaging lipid and protein peroxidation, impaired mitochondrial oxidative metabolism, and activation of cellular inflammation.25,26The effect of hyperoxia on neurologic outcomes after TBI may be similar to a focal cerebral insult; although the mechanically disrupted brain is likely unsalvageable, the surrounding vulnerable tissue may benefit from hyperoxia in the face of reduced cerebral blood flow and altered cerebral hemodynamics.11The combination of TBI and HS that occurs after polytrauma may represent a unique scenario with regard to the use of oxygen because the insult includes both TBI, which may be favorably influenced by supplemental oxygen, and the damaging effects of ischemia–reperfusion. We have developed and previously characterized a unique mouse model of controlled cortical impact (CCI) coupled with a brief, severe, pressure-controlled HS with a clinically relevant mean arterial pressure (MAP)-targeted resuscitation permitting study of acute systemic and cerebral physiology, neuropathology, and long-term functional outcomes.27We sought to determine whether treatment with oxygen versus room air (RA) influenced resuscitation and neuropathologic outcome in our combined CCI and HS mouse model. In this article, we present results showing that hyperoxia versus RA improved cerebral hemodynamics and CA3 hippocampal neuron survival but resulted in worsened oxidative stress and neuroinflammation.The Institutional Animal Care and Use Committee of the University of Pittsburgh approved all experiments. Adult (12–15 weeks of age) C57BL6 male mice (Jackson Laboratories, Bar Harbor, ME) weighing 27.7 ± 0.5 g were housed in controlled environmental conditions and allowed ad libitum access to food and water until the start of the study. We used our established mouse combined CCI plus HS model as described by Hemerka et al. 27with four experimental groups: sham RA (burr hole, brain temperature probe, catheters, anesthesia, RA during prehospital and hospital phases, but no CCI or HS), sham oxygenation (craniotomy, catheters, anesthesia, pure oxygen via nose cone during prehospital and hospital phases, but no CCI or HS), combined CCI plus HS and RA (RA administered throughout protocol), or combined CCI plus HS and oxygenation (pure oxygen via nose cone during prehospital and hospital phases). Naïve mice were also used for the various brain tissue measurements detailed below. Naïve mice were age-, sex-, and vendor-matched C57BL6 mice not exposed to any insult or therapy. Briefly, anesthesia was induced with isoflurane 4% in 2:1 N2O–O2via nose cone in spontaneous breathing mice. Maintenance of anesthesia for subsequent surgical procedures was with isoflurane 1.5% and in a 2:1 N2O–O2mixture. Femoral arterial and venous catheter access (modified PE-50 tubing) was via inguinal cutdown. Mice were placed in a stereotactic frame (David Kopf Instruments, Tujunga, CA), and a 5-mm left parietal craniotomy was performed using a dental drill with removal of bone flap. After craniotomy, anesthesia was decreased and maintained with 1% isoflurane in RA for a 10-min stabilization period. A mild to moderate CCI to the left parietal cortex was performed via a pneumatic impactor (Bimba Manufacturing Co., Monee, IL) with a flat 3-mm tip at a velocity of 5 m/s to a depth of 1 mm. The craniotomy was closed by replacing the bone flap and sealing with dental cement. Next, a 35-min pressure-controlled hemorrhagic “shock” phase (MAP, 25 ± 2 mmHg) was initiated by withdrawing blood (2.3 ml/100 g; citrated for later reinfusion) over 5 min. Continued removal or reinfusion of the autologous blood via the IV route in 0.05-ml aliquots was performed to rigorously maintain MAP of 25 ± 2 mmHg during the shock phase. The mice were allowed to spontaneously breathe 0.5% isoflurane in RA during this phase. Thirty-five minutes after CCI and HS (or at an equivalent anesthesia time point in the sham groups), spontaneously breathing mice were randomized to treatment with either oxygen (“hyperoxia”) or RA by nose cone administration upon entering the “prehospital” phase. Prehospital resuscitation consisted of an initial IV bolus of 20 ml/kg of lactated Ringer’s solution followed by maintenance lactated Ringer’s fluid at 4 ml kg-1h-1. Lactated Ringer’s boluses (10 ml/kg) were used every 5 min as needed to maintain MAP greater than 70 mmHg for the 90-min duration of prehospital resuscitation. The final “hospital” phase simulated definitive care by reinfusion of shed blood over 15 min, during which the experimental fraction of inspired oxygen was maintained and administered with 1% isoflurane. After this phase, the femoral catheters were removed, incisions were closed and infiltrated with bupivacaine 0.25% (0.05 cc), and the inhalational anesthetic was discontinued. Mice treated with oxygen were allowed to recover with supplemental oxygen, whereas those in the RA group were allowed to recover on RA. Both groups were returned to their cages after they were fully awake. Resuscitation fluid volumes administered in each phase of the experimental protocol were recorded.MAP and heart rate were monitored continuously via the femoral arterial cannula and recorded at baseline; after CCI; and every 5 min during the shock, prehospital, and hospital phases in all mice. Intracranial pressure (ICP) was similarly monitored and recorded in select mice (n = 14 that were subsequently sacrificed for histologic assessment at 7 days after injury) using a 2-French ultra-miniature pressure transducer (SPR-407; Millar, Houston, TX) inserted into the right frontal cortex (contralateral to the CCI) through a burr hole. PbtO2was also monitored and recorded in a separate group of mice (n = 8, not used for additional outcome studies) using a microelectrode (Unisense, 50 μm; Arhus, Denmark) stereotactically inserted via a burr hole through the cortex into the left dorsal hippocampus (ipsilateral to the CCI; bregma −2.5 AP, −2.0 ML; depth, 2.0 mm). Arterial blood gas measurements were obtained at baseline, at the end of the shock phase (35 min after CCI and HS), after the 90-min prehospital resuscitation (125 min after CCI and HS), and at the completion of the hospital phase (140 min after CCI and HS). Lactate, hematocrit, base deficit, and glucose values were recorded in conjunction with the arterial blood gas measurements (Stat Profile; Nova Biomedical, Waltham, MA). Temperature was monitored in the brain (via a separate burr hole in the right frontal lobe bone) and rectum, and brain temperature was controlled at 37.0 ± 0.5°C using a heat lamp throughout the experimental protocol.Brain tissue samples were obtained at 24 h after CCI and HS injuries (or the equivalent anesthesia time point in sham animals) from mice anesthetized with 4% isoflurane and euthanized by transcardial perfusion with 50 ml of ice-cold heparinized saline. Brains were removed and divided into ipsilateral and contralateral hemispheres. Cortices and hippocampi were dissected and snap frozen in liquid nitrogen, and all samples were stored at −80°C until later use.Brain samples were obtained according to methods described above at 24 h after injury from 21 mice randomized to each of the aforementioned four groups (n = 5 each from sham RA, sham oxygenation, and combined CCI plus HS and RA, and n = 6 from combined CCI plus HS and oxygenation) as well as from five naïve animals. According to methods previously reported by Belikova et al .,28the brain tissue samples were thawed on ice and homogenized using a Tissue Tearor (BioCold Scientific, Fenton, MO) in lysis buffer (tissue weight/volume ratio of 1:20) consisting of sodium chloride (0.1 M final concentration), Tris chloride (0.01 M final concentration), and Triton X (1%) at pH adjusted to 7.4. Homogenates were centrifuged at 1,000g for 10 min and then divided into aliquots for measurement of protein, ascorbate, reduced glutathione, and superoxide dismutase (SOD) activity. Metaphosphoric acid was added to a final concentration of 5% (v/v) to the aliquots for ascorbate measurements, and all samples were stored at −80 °C until use.At the time of the measurement of ascorbate, samples were thawed on ice and vortexed for 10 s every 2 min at least five times, and then centrifuged at 12,000g for 5 min at 4°C. The supernatant was collected into a microcentrifuge tube and the pH was adjusted to using 10 M in each using pH The of base for the was was divided into each the of the ascorbate, of the aliquots was treated with 1 of ascorbate for min at room all samples were they were a with ascorbate After of aliquots in was added using a to each and samples were for min at room temperature in the and using a MA). was using a ± for and a ± for from the ascorbate samples were from and to protein for with acid and were in using a MO) that a upon with was used to levels of reduced glutathione, and protein groups according to methods described upon of to brain tissue of the of according to a The after of and for min to an of the was then levels were then by the the of the and the protein levels were by 4 sodium to each and the A was used to at and of and were in was by the to the methods described by et al. was in the of 1 to both and whereas the was in the of were in and levels in brain samples obtained 24 h after injury were with a using the MAP according to the and previously described tissue supernatant from ipsilateral and contralateral hippocampi and from 20 mice randomized to each of the aforementioned four groups RA, sham oxygenation, combined CCI plus HS and RA, combined CCI plus HS and n = 5 as well as from four naïve was for levels of 21 and that the upon activation and and in the brain were with the Scientific, and all and values were of mice CCI plus HS and RA and combined CCI plus HS and n = were at 7 after injury for hippocampal neuronal The were anesthetized with 4% isoflurane and by transcardial perfusion with ice-cold followed by according to methods tissue was with and in from bregma to were from each brain and with and Scientific, neuronal survival was as as the of in and CA3 by an to treatment group using a and of as reported for and group were used to the with using for was performed group effect were stress were groups using with for using the levels were groups using with for were RA and oxygen treatment groups using the are as mean ± A of was was used for the of used in each of the study as well as the A of mice were in the combined CCI plus HS model and with either oxygen (n = or room air (n = A select of mice from each treatment group room were for measurement of (n = 6 and n = 8, and = 4 in each 24 mice were sacrificed for measurement of levels of oxidative stress and 7 the mice were sacrificed for histologic mice sacrificed at 7 days for histologic were the mice in the contralateral PbtO2was in hippocampus the cortical The measurement was obtained upon of the oxygen after the CCI injury and was ± mmHg on RA mean for all mice in both n = in mice A was obtained 5 min later ± after which HS was initiated according to the The of HS to the CCI injury reduced hippocampal an pressure of ± mmHg during the 35-min shock phase phase mean for all mice in both groups), although the to ± mmHg during the shock phase was not different than after CCI ± mmHg) or 5 min after CCI ± of mice to treatment with oxygen, however, a and in the hippocampal to treatment with RA during the prehospital phase mean prehospital phase ± ± = the hospital phase, the the and groups was to mean hospital phase ± ± = resuscitation resulted in in the prehospital and hospital phases to RA was a in pH in mice treated with oxygen versus RA during the prehospital phase (n = that likely resulted from the in the prehospital and hospital phases in versus RA groups blood levels increased and the base worsened during the shock phase, with similar in both treatment groups during the prehospital and hospital phases to and maintain the MAP mmHg) during the prehospital resuscitation phase were reduced by treatment with oxygen versus RA ± 6 ± 4 = reduced fluid the mean MAP was in the although the was not with the RA group the mean was and mean cerebral perfusion pressure was with hyperoxic resuscitation to however, the group were not and oxidative stress were by hippocampal and cortical ascorbate, reduced glutathione, and protein as well as and in samples ipsilateral and contralateral to the injury in mice at 24 h after combined CCI and was a in ascorbate levels in hippocampus of mice with oxygen versus RA (ipsilateral combined CCI and HS and oxygenation combined CCI and HS and ± ± protein, contralateral combined CCI plus HS and oxygenation combined CCI and HS and ± ± protein, = levels were ± and ± protein in left and right in naïve ipsilateral cortical ascorbate levels were in versus this was not levels were increased in the ipsilateral hippocampus of mice with RA CCI plus HS and ± to sham RA, and sham values were ± ± and ± protein, = but not to with oxygen CCI plus HS and oxygenation, ± were no groups in levels in the contralateral hippocampus or either was greater in the ipsilateral hippocampus of naïve mice and treated with RA CCI plus HS and versus both sham but the the oxygen and RA resuscitation groups was not were no in cortical reduced levels all was by protein levels in brain samples at 24 h after injury. was no effect of hyperoxic resuscitation on the of protein in ipsilateral or contralateral hippocampus and was no in the of in the hippocampus or cortex in from any was increased in the ipsilateral hippocampus of mice with oxygen versus naïve CCI plus HS and oxygenation ± ± protein, = but not to RA resuscitation CCI plus HS and RA, ± in the cortex was not different In ascorbate levels in the hippocampus were reduced in the versus was no in cortical ascorbate levels in the oxygen versus RA was also no effect of oxygen on the levels of reduced glutathione, protein or in the hippocampus or cortex to RA in mice at 24 h after combined CCI and of and a of with regard to the effect of combined CCI and HS and the impact of treatment with supplemental were and were increased in hippocampus versus either naïve or sham groups The oxygen and RA treatment groups was for the with levels seen in hippocampus after In the five of the were increased versus either naïve or sham The of the in cortex was similar to that seen in The treatment groups in the however, was a in with RA with the hyperoxia were increased in brain at 24 h after combined CCI plus but was the by hyperoxia versus RA treatment with to oxygen in the hippocampus and cortex were and In five were increased after injury versus either naïve or sham groups and was increased in hippocampus of both treatment and were increased in hippocampus in the whereas and were increased in hippocampus in the RA In were increased versus either naïve or sham and in and were seen after injury in the RA was no in cortical levels with regard to oxygen or RA The effects of oxygen on was an in hippocampal and whereas hippocampal and and cortical and were decreased with RA the of with were and treatment were and In was increased in hippocampus after injury versus either naïve or sham groups The in hippocampal was greater in the oxygen group with the RA and were not increased in hippocampus after combined CCI and HS injury. In and were increased after injury versus either naïve or sham The in cortical after CCI and HS was greater in the RA versus oxygen however, was no effect of oxygen on cortical versus RA. in cortex was not increased versus naïve or sham after combined CCI plus The effect of oxygen on the after combined CCI plus HS injury was an in hippocampal and in cortical with RA in the vulnerable and CA3 hippocampal the cortical were in and at 7 days after combined CCI and HS and resuscitation. was a greater of in the CA3 of mice with oxygen versus RA ± 2 15 ± 2 CA3 was also a improved survival of in the however, the from RA was not = resuscitation after combined CCI and HS in mice reduced fluid and CA3 hippocampal neuronal a vulnerable brain in our an in oxidative stress as by ascorbate and a a in brain versus RA. to the with oxygen administration after cardiac combined CCI and HS a scenario supplemental oxygen is those in studies of also of to maintain a after the in TBI that oxygen may have study is used a mouse model CCI with a pressure-controlled HS followed by resuscitation. the prehospital scenario supplemental oxygen be as by for prehospital treatment of into that the not be of pure oxygen via nose cone a greater than in the RA. The increased with oxygen versus RA We were by the to oxygen, that the additional oxygen is in the that oxygen use after combined CCI and HS in our model reduced resuscitation fluid to the MAP mmHg) during the prehospital phase. systemic because of a in systemic is by a in cardiac secondary to a in heart shock hyperoxia hemodynamics and blood flow to results showed that reduced fluid oxygen administration increased mean MAP versus RA, although the in MAP was not to a MAP the clinical scenario to cerebral perfusion pressure during resuscitation to secondary is not a effects according to the and the 100% oxygen cerebral that is by an in increased oxygen oxygen administration in with TBI improves and cerebral that oxygen administration in mice a mean and increased mean cerebral perfusion pressure after combined CCI and however, were not different from RA pH decreased and in mice treated with oxygen versus RA. The in pH and in resulted from of the oxygen not however, we the to oxygen in separate mice and a decreased rate versus RA not resulted from decreased in the oxygen group as reported in is in our study that we a but not different mean during resuscitation with oxygen versus RA a is and brain are decreased after with our hyperoxia this brain after combined CCI and HS versus RA. decreased in the hippocampus and contralateral to the cortical at 24 h in mice with is the of oxidative and reduced levels represent a of oxidative in hippocampal ascorbate with hyperoxia is but may be the of the in hippocampal neuronal survival is the of resuscitation with oxygen versus RA. The neuroprotective effects of oxygen resuscitation be improved by combination of 100% oxygen with an in combined CCI and We not a of ascorbate in the although was a mean cortical ascorbate with oxygen treatment versus RA. The cortex is after the of CCI perfusion and oxygen we not cortical In of of oxidative we not in levels of reduced glutathione, or after resuscitation with oxygen versus RA. may be that the effect of hyperoxia on brain is not as as or that the duration of oxygen was We an in in the hippocampus of mice with oxygen versus but not versus RA. is the of superoxide in and is induced after in hippocampus is our showing of by neuronal after plus HS likely a unique study on our showing after combined CCI and HS in mice by and in and hippocampal and cortical tissue in the in are likely to the impact of oxygen represent the after combined CCI and HS in mouse brain and cellular oxygen reduced neuronal death but increased oxidative could not a that oxygen or tissue and oxidative with our was a in levels after combined CCI and although oxygen not to an of of oxygen on however, effects that are the effect of oxygen on was an in hippocampal and whereas hippocampal and and cortical and were decreased versus RA. and are after TBI, with increased in brain that it is a TBI whether of increased in hippocampus after combined CCI and HS is or is that is a effect of is also in and the of after oxygen not oxygen in and in cortex an in by a neuroprotective in has may our also a effect of supplemental also a in in in hippocampus were also by is a for oxygen the cortical after combined CCI and the of tissue in and the in oxygen in our this is not The impact of this however, is and could either a in or of increased evidence of oxidative stress in oxygen administration after combined CCI and HS hippocampal CA3 neuronal death at 7 days after injury. The moderate CCI we used was to the hippocampus but vulnerable to were to benefit from oxygen on CA3 than as we that the hippocampus the cortical injury a We a in with oxygen a is needed to our in in it vulnerable to secondary which benefit from oxygen during TBI plus HS may and effects of is similar to benefit was seen during but effects during study has TBI plus HS is a scenario with We used a injury a of the could on the clinical our model includes a clinically relevant injury and resuscitation. We administered pure oxygen to the effect of but it is of the fraction of inspired oxygen We also male which are vulnerable to oxidative stress than of and long-term outcomes are also the supplemental oxygen use after cardiac and TBI, we sought to determine whether oxygen influenced resuscitation and neuropathologic outcome in TBI plus improved cerebral hemodynamics and CA3 hippocampal neuron survival but worsened oxidative stress versus RA and increased The benefit of hyperoxic resuscitation the to the of an to supplemental oxygen or other to oxygen after TBI plus HS