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

Neonatal systemic inflammation induces inflammatory reactions and brain apoptosis in a pathogen-specific manner

&lt;b&gt;&lt;i&gt;Background:&lt;/i&gt;&lt;/b&gt; 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 (PAM&lt;sub&gt;3&lt;/sub&gt;CSK&lt;sub&gt;4&lt;/sub&gt; [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. &lt;b&gt;&lt;i&gt;Objective:&lt;/i&gt;&lt;/b&gt; We aimed to examine whether systemic gram-positive or gram-negative inflammatory sensitisation affects juvenile rat pups per se, without an HI insult. &lt;b&gt;&lt;i&gt;Methods:&lt;/i&gt;&lt;/b&gt; Neonatal 7-day-old rats (&lt;i&gt;n&lt;/i&gt; = 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-1β, TNF-α, and IL-10, via PCR), apoptosis (cleaved caspase 3, via Western blots), and microglial activation (Iba1, via immunohistochemistry) were examined. &lt;b&gt;&lt;i&gt;Results:&lt;/i&gt;&lt;/b&gt; 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. &lt;b&gt;&lt;i&gt;Conclusion:&lt;/i&gt;&lt;/b&gt; 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.</jats:p

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

&lt;b&gt;&lt;i&gt;Introduction:&lt;/i&gt;&lt;/b&gt; 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. &lt;b&gt;&lt;i&gt;Material and Methods:&lt;/i&gt;&lt;/b&gt; 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. &lt;b&gt;&lt;i&gt;Results:&lt;/i&gt;&lt;/b&gt; 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 &lt; 0.001). Additionally, a significant increase in astrogliosis (glial fibrillary acidic expression, p &lt; 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. &lt;b&gt;&lt;i&gt;Conclusion:&lt;/i&gt;&lt;/b&gt; 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.</jats:p

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

Background: Pre-clinical research on the neuroprotective effect of hypothermia after perinatal asphyxia has shown variable results, depending on co-morbidities 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 pre-sensitisation with lipopolysaccharide (LPS) prior to a mild HI insult negates hypothermic neuroprotection. However, the neuroprotective effect of HT is fully maintained after gram-positive type pre-sensitisation with PAM3CSK4 (PAM) in the same HI model. Whether HT is neuroprotective in severe brain injury with gram-positive inflammatory pre-sensitisation 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 dditional 196 pups received intraperitoneal 0.9% saline (control) or PAM1mg/kg, 8 h before undergoing the same HI insult. After randomisation to 5 h normothermia (NT37°C) or HT32°C, pups survived one week before they were sacrificed by perfusion fixation. Brains were harvested for hemispheric (HEM) and ippocampal (HIP) area loss analyses at P14, as well as immunostaining for neuron count in the HIP CA1 region. Results: Normothermic PAM animals (PAM-NT) had a comparable median area loss (HEM: 60% (95% CI 33-66); HIP: 61% (95% CI 29-67)) to vehicle animals (Veh-NT) (HEM: 58% (95% CI 11-64); HIP: 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%). HT significantly reduced HEM and HIP injury in the Veh group (HEM: p=0.048; HIP: p=0.042) as well as in the PAM group (HEM: p=0.03; HIP: p=0.027). Conclusion: In these experiments with severe brain injury, TLR-2 exposure prior to HI does not have an additive injurious effect, and there is a small but significant neuroprotective effect of HT. Hypothermia 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-1positive inflammatory exposure prior to HI injury does not negate a neuroprotective effect of HT in severe brain injury.<br/