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.

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.

This randomized, double-blind, crossover study in two parts compared tolerability to high doses of formoterol (Oxis Turbuhaler) with that of high doses of terbutaline (Bricanyl Turbuhaler). After Holter monitoring at home, 12 patients were treated with 4+4+4 doses of formoterol Turbuhaler, 6 microg x dose(-1), (total daily metered dose 72 microg) or 4+4+4 doses of terbutaline Turbuhaler, 0.5 mg x dose(-1) (daily dose 6 mg) given in the morning, after lunch and in the evening, for 3 consecutive days. After a one week washout period at home, patients received the alternative treatment. Thereafter, 15 other patients received 8+6+6 doses of formoterol Turbuhaler (total daily metered dose 120 microg) or 8+6+6 doses of terbutaline Turbuhaler (daily dose 10 mg). Pulse, cardiac frequency, blood pressure, serum potassium, electrocardiogram and forced expiratory volume in one second (FEV1) were registered at regular intervals and Holter monitoring was applied during all 4 treatment days. Terbutaline 6 mg showed significantly greater systemic effects than formoterol 72 microg on pulse, blood pressure, cardiac frequency and QTc (QT interval corrected for heart rate). Terbutaline 10 mg had significantly greater effects than formoterol 120 microg on serum potassium levels, pulse, cardiac frequency and QTc. No differences in FEV1 levels were found. Both drugs were safe and generally well tolerated on both dose levels. In conclusion, high doses of formoterol Turbuhaler over 3 days were generally safe and well tolerated. Daily doses of 6 mg and 10 mg terbutaline Turbuhaler were systemically more potent than 72 microg and 120 microg formoterol, respectively. The safety margin thus appears to be wide if patients happen to use extra doses of formoterol in addition to those prescribed for regular use.

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.

Therapeutic hypothermia (HT) is standard care for term infants with hypoxic-ischaemic (HI) encephalopathy. However, the efficacy of HT in preclinical models, such as the Vannucci model of unilateral HI in the newborn rat, is often greater than that reported from clinical trials. Here, we report a meta-analysis of data from every experiment in a single laboratory, including pilot data, examining the effect of HT in the Vannucci model. Across 21 experiments using 106 litters, median (95% CI) hemispheric area loss was 50.1% (46.0-51.9%; n = 305) in the normothermia group, and 41.3% (35.1-44.9%; n = 317) in the HT group, with a bimodal injury distribution. Median neuroprotection by HT was 17.6% (6.8-28.3%), including in severe injury, but was highly-variable across experiments. Neuroprotection was significant in females (p < 0.001), with a non-significant benefit in males (p = 0.07). Animals representing the median injury in each group within each litter (n = 277, 44.5%) were also analysed using formal neuropathology, which showed neuroprotection by HT throughout the brain, particularly in females. Our results suggest an inherent variability and sex-dependence of the neuroprotective response to HT, with the majority of studies in the Vannucci model vastly underpowered to detect true treatment effects due to the distribution of injury. The use of animal research has dramatically advanced human knowledge of physiology, pathology, and sciencebased medicine 1. However, despite centuries of improvement in the methodological and ethical approaches to experiments involving animals 1 , preclinical research across all fields of medicine has also significantly underperformed with regards to the translation of robust treatments for complex diseases in humans 2. For perinatal asphyxia and subsequent hypoxic-ischaemic encephalopathy (HIE), just one treatment so far, therapeutic hypothermia (HT), has emerged from the preclinical research to provide a robust treatment effect in term newborns with moderate-to-severe neurological injury 3. A recent meta-analysis of those clinical trials found that the numbers needed to treat (NNT) were 11 to reduce mortality, and 8 to reduce major neurodevelopmental disability 3. However, HT is not universally neuroprotective. Meta-analyses of the original large clinical trials of HT found that 40-50% of treated infants still experienced a poor outcome (death or severe disability) 4 , though more recent trials suggest it is now around 30% 5. In the most widely-researched model of neonatal hypoxic-ischaemic (HI) brain injury, the Vannucci model of unilateral HI, our group and others have traditionally found that HT provides around 40% reduction in neuropathology score and tissue loss 6-9. This neuroprotective effect of HT was corroborated in larger animal models 10-12 ,