Hypothermia Is Neuroprotective after Severe Hypoxic-Ischaemic Brain Injury in Neonatal Rats Pre-Exposed to PAM3CSK4.

BACKGROUND: Preclinical research on the neuroprotective effect of hypothermia (HT) after perinatal asphyxia has shown variable results, depending on comorbidities and insult severity. Exposure to inflammation increases vulnerability of the neonatal brain to hypoxic-ischaemic (HI) injury, and could be one explanation for those neonates whose injury is unexpectedly severe. Gram-negative type inflammatory exposure by lipopolysaccharide administration prior to a mild HI insult results in moderate brain injury, and hypothermic neuroprotection is negated. However, the neuroprotective effect of HT is fully maintained after gram-positive type inflammatory exposure by PAM3CSK4 (PAM) pre-administration in the same HI model. Whether HT is neuroprotective in severe brain injury with gram-positive inflammatory pre-exposure has not been investigated. METHODS: 59 seven-day-old rat pups were subjected to a unilateral HI insult, with left carotid artery ligation followed by 90-min hypoxia (8% O2 at Trectal 36°C). An additional 196 pups received intraperitoneal 0.9% saline (control) or PAM1 mg/kg, 8 h before undergoing the same HI insult. After randomisation to 5 h normothermia (NT37°C) or HT32°C, pups survived 1 week before they were sacrificed by perfusion fixation. Brains were harvested for hemispheric and hippocampal area loss analyses at postnatal day 14, as well as immunostaining for neuron count in the HIP CA1 region. RESULTS: Normothermic PAM animals (PAM-NT) had a comparable median area loss (hemispheric: 60% [95% CI 33-66]; hippocampal: 61% [95% CI 29-67]) to vehicle animals (Veh-NT) (hemispheric: 58% [95% CI 11-64]; hippocampal: 60% [95% CI 19-68]), which is defined as severe brain injury. Furthermore, mortality was low and similar in the two groups (Veh-NT 4.5% vs. PAM-NT 6.6%). HT reduced hemispheric and hippocampal injury in the Veh group by 13 and 28%, respectively (hemispheric: p = 0.048; hippocampal: p = 0.042). HT also provided neuroprotection in the PAM group, reducing hemispheric injury by 22% (p = 0.03) and hippocampal injury by 37% (p = 0.027). CONCLUSION: In these experiments with severe brain injury, Toll-like receptor-2 triggering prior to HI injury does not have an additive injurious effect, and there is a small but significant neuroprotective effect of HT. HT appears to be neuroprotective over a continuum of injury severity in this model, and the effect size tapers off with increasing area loss. Our results indicate that gram-positive inflammatory exposure prior to HI injury does not negate the neuroprotective effect of HT in severe brain injury.

Neonatal Systemic Inflammation Induces Inflammatory Reactions and Brain Apoptosis in a Pathogen-Specific Manner.

BACKGROUND: After neonatal asphyxia, therapeutic hypothermia (HT) is the only proven treatment option. Although established as a neuroprotective therapy, benefit from HT has been questioned when infection is a comorbidity to hypoxic-ischaemic (HI) brain injury. Gram-negative and gram-positive species activate the immune system through different pathogen recognition receptors and subsequent immunological systems. In rodent models, gram-negative (lipopolysaccharide [LPS]) and gram-positive (PAM3CSK4 [PAM]) inflammation similarly increase neuronal vulnerability to HI. Interestingly, while LPS pre-sensitisation negates the neuroprotective effect of HT, HT is highly beneficial after PAM-sensitised HI brain injury. OBJECTIVE: We aimed to examine whether systemic gram-positive or gram-negative inflammatory sensitisation affects juvenile rat pups per se, without an HI insult. METHODS: Neonatal 7-day-old rats (n = 215) received intraperitoneal injections of vehicle (0.9% NaCl), LPS (0.1 mg/kg), or PAM (1 mg/kg). Core temperature and weight gain were monitored. Brain cytokine expression (IL-6, IL-1ß, TNF-α, and IL-10, via PCR), apoptosis (cleaved caspase 3, via Western blots), and microglial activation (Iba1, via immunohistochemistry) were examined. RESULTS: LPS induced an immediate drop in core temperature followed by poor weight gain, none of which were seen after PAM. Furthermore, LPS induced brain apoptosis, while PAM did not. The magnitude and temporal profile of brain cytokine expression differed between LPS- and PAM-injected animals. CONCLUSION: These findings reveal sepsis-like conditions and neuroinflammation specific to the inflammatory stimulus (gram-positive vs. gram-negative) in the neonatal rat. They emphasise the importance of pre-clinical models being pathogen dependent, and should always be carefully tailored to their clinical scenario.

Hypothermic Neuronal Rescue from Infection-Sensitised Hypoxic-Ischaemic Brain Injury Is Pathogen Dependent.

Perinatal infection increases the vulnerability of the neonatal brain to hypoxic-ischaemic (HI) injury. Hypothermia treatment (HT) does not provide neuroprotection after pre-insult inflammatory sensitisation by lipopolysaccharide (LPS), a gram-negative bacterial wall constituent. However, early-onset sepsis in term babies is caused by gram-positive species in more than 90% of cases, and neuro-inflammatory responses triggered through the gram-negative route (Toll-like receptor 4, TLR-4) are different from those induced through the gram-positive route via TLR-2. Whether gram-positive septicaemia sensitises the neonatal brain to hypoxia and inhibits the neuroprotective effect of HT is unknown. Seven-day-old Wistar rats (n = 178) were subjected to intraperitoneal injections of PAM3CSK4 (1 mg/kg, a synthetic TLR-2 agonist) or vehicle (0.9% NaCl). After an 8-h delay, the left carotid artery was ligated followed by 50 min of hypoxia (8% O2) at a rectal temperature of 36°C. Pups received a 5-h treatment of normothermia (NT, 37°C) or HT (32°C) immediately after the insult. Brains were harvested after 7 days' survival for hemispheric and hippocampal area loss analyses and immunolabelling of microglia (Iba1) and hippocampal neurons (NeuN). Normothermic PAM3CSK4-injected animals showed significantly more brain injury than vehicle animals (p = 0.014). Compared to NT, HT significantly reduced injury in the PAM3CSK4-injected animals, with reduced area loss (p < 0.001), reduced microglial activation (p = 0.006), and increased neuronal rescue in the CA1 region (p < 0.001). Experimental induction of a sepsis-like condition through the gram-positive pathway sensitises the brain to HI injury. HT was highly neuroprotective after the PAM3CSK4-triggered injury, suggesting HT may be neuroprotective in the presence of a gram-positive infection. These results are in strong contrast to LPS studies where HT is not neuroprotective.

Inhibition of Constitutive Nitric Oxide Synthase Does Not Influence Ventilation-Perfusion Matching in Normal Prone Adult Sheep With Mechanical Ventilation.

BACKGROUND: Local formation of nitric oxide in the lung induces vasodilation in proportion to ventilation and is a putative mechanism behind ventilation-perfusion matching. We hypothesized that regional ventilation-perfusion matching occurs in part due to local constitutive nitric oxide formation. METHODS: Ventilation and perfusion were analyzed in lung regions (≈1.5 cm) before and after inhibition of constitutive nitric oxide synthase with N-nitro-L-arginine methyl ester (L-NAME) (25 mg/kg) in 7 prone sheep ventilated with 10 cm H2O positive end-expiratory pressure. Ventilation and perfusion were measured by the use of aerosolized fluorescent and infused radiolabeled microspheres, respectively. The animals were exsanguinated while deeply anesthetized; then, lungs were excised, dried at total lung capacity, and divided into cube units. The spatial location for each cube was tracked and fluorescence and radioactivity per unit weight determined. RESULTS: After administration of L-NAME, pulmonary artery pressure increased from a mean of 16.6-23.6 mm Hg, P = .007 but PaO2, PaCO2, and SD log(V/Q) did not change. Distribution of ventilation was not influenced by L-NAME, but a small redistribution of perfusion from ventral to dorsal lung regions was observed. Perfusion to regions with the highest ventilation (fifth quintile of the ventilation distribution) remained unchanged after L-NAME. CONCLUSIONS: We found minimal or no influence of constitutive nitric oxide synthase inhibition by L-NAME on the distributions of ventilation and perfusion, and ventilation-perfusion in prone, anesthetized, ventilated, and healthy adult sheep with normal gas exchange.

Treatment temperature and insult severity influence the neuroprotective effects of therapeutic hypothermia.

Therapeutic hypothermia (HT) is standard care for moderate and severe neonatal hypoxic-ischaemic encephalopathy (HIE), the leading cause of permanent brain injury in term newborns. However, the optimal temperature for HT is still unknown, and few preclinical studies have compared multiple HT treatment temperatures. Additionally, HT may not benefit infants with severe encephalopathy. In a neonatal rat model of unilateral hypoxia-ischaemia (HI), the effect of five different HT temperatures was investigated after either moderate or severe injury. At postnatal-day seven, rat pups underwent moderate or severe HI followed by 5 h at normothermia (37 °C), or one of five HT temperatures: 33.5 °C, 32 °C, 30 °C, 26 °C, and 18 °C. One week after treatment, neuropathological analysis of hemispheric and hippocampal area loss, and CA1 hippocampal pyramidal neuron count, was performed. After moderate injury, a significant reduction in hemispheric and hippocampal loss on the injured side, and preservation of CA1 pyramidal neurons, was seen in the 33.5 °C, 32 °C, and 30 °C groups. Cooling below 33.5 °C did not provide additional neuroprotection. Regardless of treatment temperature, HT was not neuroprotective in the severe HI model. Based on these findings, and previous experience translating preclinical studies into clinical application, we propose that milder cooling should be considered for future clinical trials.

Hypothermia Does Not Reverse Cellular Responses Caused by Lipopolysaccharide in Neonatal Hypoxic-Ischaemic Brain Injury.

INTRODUCTION: Bacterial lipopolysaccharide (LPS) injection prior to hypoxia-ischaemia significantly increases hypoxia-ischaemic brain injury in 7-day-old (P7) rats. In addition, therapeutic hypothermia (HT) is not neuroprotective in this setting. However, the mechanistic aspects of this therapeutic failure have yet to be elucidated. This study was designed to investigate the underlying cellular mechanisms in this double-hit model of infection-sensitised hypoxia-ischaemic brain injury. MATERIAL AND METHODS: P7 rat pups were injected with either vehicle or LPS, and after a 4-hour delay were exposed to left carotid ligation followed by global hypoxia inducing a unilateral stroke-like hypoxia-ischaemic injury. Pups were randomised to the following treatments: (1) vehicle-treated pups receiving normothermia treatment (NT) (Veh-NT; n = 40), (2) LPS-treated pups receiving NT treatment (LPS-NT; n = 40), (3) vehicle-treated pups receiving HT treatment (Veh-HT; n = 38) and (4) LPS-treated pups receiving HT treatment (LPS-HT; n = 35). On postnatal day 8 or 14, Western blot analysis or immunohistochemistry was performed to examine neuronal death, apoptosis, astrogliosis and microglial activation. RESULTS: LPS sensitisation prior to hypoxia-ischaemia significantly exacerbated apoptotic neuronal loss. NeuN, a neuronal biomarker, was significantly reduced in the LPS-NT and LPS-HT groups (p = 0.008). Caspase-3 activation was significantly increased in the LPS-sensitised groups (p < 0.001). Additionally, a significant increase in astrogliosis (glial fibrillary acidic expression, p < 0.001) was seen, as well as a trend towards increased microglial activation (Iba 1 expression, p = 0.051) in LPS-sensitised animals. Treatment with HT did not counteract these changes. CONCLUSION: LPS-sensitised hypoxia-ischaemic brain injury in newborn rats is mediated through neuronal death, apoptosis, astrogliosis and microglial activation. In this double-hit model, treatment with HT does not ameliorate these changes.

Hypothermia is not neuroprotective after infection-sensitized neonatal hypoxic-ischemic brain injury.

BACKGROUND: Therapeutic hypothermia (HT) is the standard treatment after perinatal hypoxic-ischemic (HI) injury. Infection increases vulnerability to HI injury, but the effect of HT on lipopolysaccharide (LPS) sensitized HI brain injury is unknown. DESIGN/METHODS: P7 rat pups were injected either with vehicle or LPS, and after a 4h delay they were exposed to left carotid ligation followed by global hypoxia inducing a unilateral stroke-like HI injury. Pups were randomized to the following treatments: (1) vehicle treated HI-pups receiving normothermia treatment (NT) (Veh-NT; n=30); (2) LPS treated HI-pups receiving NT treatment (LPS-NT; n=35); (3) vehicle treated HI-pups receiving HT treatment (Veh-HT; n=29); or (4) LPS treated HI-pups receiving HT treatment (LPS-HT; n=46). Relative area loss of the left/right hemisphere and the areas of hippocampi were measured at P14. RESULTS: Mean brain area loss in the Veh-NT group was 11.2±14%. The brain area loss in LPS-NT pups was 29.8±17%, which was significantly higher than in the Veh-NT group (p=0.002). The Veh-HT group had a significantly smaller brain area loss (5.4±6%), when compared to Veh-NT group (p=0.043). The LPS-HT group showed a brain area loss of 32.5±16%, which was significantly higher than in the Veh-HT group (p<0.001). LPS-HT group also had significantly smaller size of the left hippocampus, which was not found in other groups. LPS-sensitization significantly decreased the sizes of the right, unligated-hemispheres, independent of post-HI treatment. CONCLUSIONS: Therapeutic hypothermia is not neuroprotective in this LPS-sensitized unilateral stroke-like HI brain injury model in newborn rats. Lack of neuroprotection was particularly seen in the hippocampus. Pre-insult exposure to LPS also induced brain area loss in the unligated hemisphere, which is normally not affected in this model.

Effects of exercise on leukocytosis and blood hemostasis in 800 healthy young females and males.

AIM: To investigate the effects of exercise on healthy individuals of both genders. METHODS: This study lasted 6 years and involved about 800 healthy people. Individuals were divided into females and males and further sub-divided into two groups; in the first group individuals run (or skied in the winter time) and then rested for 3 h, whereas individuals in the second group intensely cycled for 5 min. The status of health was determined by measuring the sedimentation rate and the intensity of exercises by measuring the heart rate. Blood samples were collected before and after exercise. RESULTS: We observed that in the first group a significant increase of the total white blood cells, segmented neutrophils, band neutrophils, eosinophils and to a lesser extent lymphocytes but not monocytes in the blood circulation. However, all cell types were increased in the circulation after 5 min intense exercise. No differences in the pattern of cell increase were observed among the genders. Activated partial thromboplastin time (APTT) and D-dimer were also measured in the blood of individuals who cycled intensely for 5 min to determine the coagulation and fibrinolytic activities in the blood. APTT is reduced and D-dimer values significantly increased after intense exercise. However, APTT was statistically lower in males than females, whereas no differences in the D-dimer values were observed among the genders. CONCLUSION: Our results indicate that exercise whether leisure or strenuous affects leukocytosis and hemostasis in both genders. A major advantage of this study is the high numbers of individuals involved and the inclusion of both females and males values.

Myocardial protection evoked by hyperoxic exposure involves signaling through nitric oxide and mitogen activated protein kinases.

BACKGROUND: Hyperoxic exposure in vivo (> 95% oxygen) attenuates ischemia-reperfusion injury, but the signaling mechanisms of this cardioprotection are not fully determined. We studied a possible role of nitric oxide (NO) and mitogen activated protein kinases (MAPK) in hyperoxic protection. METHODS: Mice (n = 7-9 in each group) were kept in normoxic or hyperoxic environments for 15 min prior to harvesting the heart and Langendorff perfusion with global ischemia (45 min) and reperfusion (60 min). Endpoints were cardiac function and infarct size. Additional hearts were collected to evaluate MAPK phosphorylation (immunoblot). The nitric oxide synthase inhibitor L-NAME, the ERK1/2 inhibitor PD98059 and the p38 MAPK inhibitor FR167653 were injected intraperitoneally before hyperoxia or normoxia. RESULTS: Hyperoxia improved postischemic functional recovery and reduced infarct size (p < 0.05). Hyperoxic exposure caused cardiac phosphorylation of the MAPK family members p38 and ERK1/2, but not JNK. L-NAME, PD98059 and FR167653 all reduced the protection afforded by hyperoxic exposure, but did not influence performance or infarction in hearts of normoxic mice. The hyperoxia-induced phosphorylation of ERK1/2 and p38 was reduced by L-NAME and both MAPK inhibitors. CONCLUSION: Nitric oxide triggers hyperoxic protection, and ERK1/2 and p38 MAPK are involved in signaling of protection against ischemia-reperfusion injury.

Marked differences between prone and supine sheep in effect of PEEP on perfusion distribution in zone II lung.

The classic four-zone model of lung blood flow distribution has been questioned. We asked whether the effect of positive end-expiratory pressure (PEEP) is different between the prone and supine position for lung tissue in the same zonal condition. Anesthetized and mechanically ventilated prone (n = 6) and supine (n = 5) sheep were studied at 0, 10, and 20 cm H2O PEEP. Perfusion was measured with intravenous infusion of radiolabeled 15-microm microspheres. The right lung was dried at total lung capacity and diced into pieces (approximately 1.5 cm3), keeping track of the spatial location of each piece. Radioactivity per unit weight was determined and normalized to the mean value for each condition and animal. In the supine posture, perfusion to nondependent lung regions decreased with little relative perfusion in nondependent horizontal lung planes at 10 and 20 cm H2O PEEP. In the prone position, the effect of PEEP was markedly different with substantial perfusion remaining in nondependent lung regions and even increasing in these regions with 20 cm H2O PEEP. Vertical blood flow gradients in zone II lung were large in supine, but surprisingly absent in prone, animals. Isogravitational perfusion heterogeneity was smaller in prone than in supine animals at all PEEP levels. Redistribution of pulmonary perfusion by PEEP ventilation in supine was largely as predicted by the zonal model in marked contrast to the findings in prone. The differences between postures in blood flow distribution within zone II strongly indicate that factors in addition to pulmonary arterial, venous, and alveolar pressure play important roles in determining perfusion distribution in the in situ lung. We suggest that regional variation in lung volume through the effect on vascular resistance is one such factor and that chest wall conformation and thoracic contents determine regional lung volume.

Positive end-expiratory pressure affects regional redistribution of ventilation differently in prone and supine sheep.

OBJECTIVE: To examine interactions between positive end-expiratory pressure (PEEP) and posture on regional distribution of ventilation and to compare measurements of regional ventilation with two aerosols: a wet fluorescent microsphere aerosol (FMS, median mass aerodynamic diameter 1.1 microm) and a dry Tc-labeled carbon particle aerosol (Technegas, TG, median mass aerodynamic diameter approximately 0.1 microm). DESIGN: Experimental study. SETTING: Academic laboratory. SUBJECTS: : Anesthetized and mechanically ventilated sheep (n = 16). INTERVENTIONS: Four conditions were studied: prone or supine posture with or without 10 cm H2O PEEP. MEASUREMENTS AND MAIN RESULTS: Comparisons of FMS and TG were made in five animals. The median correlation coefficient of the two ventilation tracers was .95 (range, .91-.96). The mean ventilation per unit weight of dry lung for horizontal planes was almost identical whether measured with TG or FMS. The distribution of ventilation was assessed by analyzing deposition of aerosol in about 1,000 lung regions per animal. Distribution of ventilation down the vertical axis was linear in prone (the slope indicated a dorsal-to-ventral three-fold difference in ventilation) but unimodal in supine animals with the mode in the center of the lung. Redistribution of ventilation with 10 PEEP differed between posture, shifting the mode in supine toward dependent lung regions while eliminating the dorsal-to-ventral gradient in prone. The regional heterogeneity in ventilation was greater in supine sheep at both levels of PEEP, and this was due mostly to greater isogravitational heterogeneity in supine than in prone position. CONCLUSIONS: The wet fluorescent microsphere aerosol was as reliable as Technegas for high-resolution measurements of regional ventilation. The markedly different effects of 10 PEEP in supine and prone sheep may have important implications for gas exchange both in noninjured and injured lungs.

Regulation of arterial blood pressure in humans during isometric muscle contraction and lower body negative pressure.

Previous studies have shown that the blood pressure response to isometric handgrip remains unchanged during reductions in preload induced by lower body negative pressure (LBNP). The purpose of the present study was to assess the beat-by-beat haemodynamic mechanisms allowing for precise control of mean arterial pressure (MAP). We have followed the cardiovascular variables involved in the regulation of MAP during isometric handgrip with and without additional application of LBNP during defined periods of the ongoing contraction. Sixteen subjects participated. Mean arterial blood pressure (MAP), heart rate (HR), stroke volume (SV), cardiac output (CO), blood flow velocity in the brachial artery, acral skin blood flow, as well as total (TPR) and local (LPR) peripheral resistance were continuously recorded/calculated before, during and after 2 min of handgrip both with and without concomitant LBNP. The main finding was that MAP increased at the same rate and to the same absolute level whether or not LBNP was applied. A uniform increase in MAP was observed even though the cardiovascular variables evolved differently in the periods with and without LBNP. At the onset of LBNP at -20 mmHg, there was a transient drop in MAP and a transient increase in HR, but within seconds, MAP was regulated back to the slope caused by the isometric handgrip proper. CO and SV, which were declining gradually, showed an additional marked but gradual reduction upon LBNP application. At the same time, both LPR and TPR increased markedly and continuously. In summary, the increase in MAP during isometric handgrip remained essentially unchanged by LBNP-induced alterations in preload. The increase in MAP was caused by a marked increase in peripheral resistance. This supports the concept of a central set point, continuously regulated upwards as long as the isometric handgrip persists. Furthermore, it reveals a considerable flexibility in the cardiovascular control mechanisms used to achieve the desired arterial pressure.

Onset of mild lower body negative pressure induces transient change in mean arterial pressure in humans.

Mild (0 to -20 mmHg) lower body negative pressure (LBNP) has traditionally been considered to elicit reflex responses mediated by cardiopulmonary baroreceptors only, without any arterial baroreflex involvement. Mild LBNP has therefore frequently been used to study the influence of cardiopulmonary baroreceptors on the human circulatory system. In a previous study we found that mean arterial pressure (MAP) was transiently but strongly affected by rapid (0.3 s) onset and release of -20 mmHg LBNP. In the present study we tested whether MAP is also transiently affected by slow onset and release of -20 mmHg LBNP. A group of 12 subjects participated in this study, which was approved by the local Ethics Committee. Heart rate, stroke volume, cardiac output, MAP, total peripheral resistance, acral and non-acral skin blood flow, and blood flow velocity in the brachial artery were continuously recorded during the pre-LBNP period, during LBNP and during the post-LBNP period. The LBNP was gradually applied and released over a 15 s period. The main finding was that MAP was transiently but strongly affected by the gradual onset of LBNP as mild as -20 mmHg. During onset of LBNP MAP was significantly ( P=0.003) lower than MAP in the pre-LBNP period. This shows that not only the cardiopulmonary baroreceptors but also the arterial baroreceptors must be activated during mild LBNP.