Marius Widerøe

Preconditioning Triggered by Carbon Monoxide (CO) Provides Neuronal Protection Following Perinatal Hypoxia-Ischemia

Perinatal hypoxia-ischemia is a major cause of acute mortality in newborns and cognitive and motor impairments in children. Cerebral hypoxia-ischemia leads to excitotoxicity and necrotic and apoptotic cell death, in which mitochondria play a major role. Increased resistance against major damage can be achieved by preconditioning triggered by subtle insults. CO, a toxic molecule that is also generated endogenously, may have a role in preconditioning as low doses can protect against inflammation and apoptosis. In this study, the role of CO-induced preconditioning on neurons was addressed in vitro and in vivo. The effect of 1 h of CO treatment on neuronal death (plasmatic membrane permeabilization and chromatin condensation) and bcl-2 expression was studied in cerebellar granule cells undergoing to glutamate-induced apoptosis. COs role was studied in vivo in the Rice-Vannucci model of neonatal hypoxia-ischemia (common carotid artery ligature +75 min at 8% oxygen). Apoptotic cells, assessed by Nissl staining were counted with a stereological approach and cleaved caspase 3-positive profiles in the hippocampus were assessed. Apoptotic hallmarks were analyzed in hippocampal extracts by Western Blot. CO inhibited excitotoxicity-induced cell death and increased Bcl-2 mRNA in primary cultures of neurons. In vivo, CO prevented hypoxia-ischemia induced apoptosis in the hippocampus, limited cytochrome c released from mitochondria and reduced activation of caspase-3. Still, Bcl-2 protein levels were higher in hippocampus of CO pre-treated rat pups. Our results show that CO preconditioning elicits a molecular cascade that limits neuronal apoptosis. This could represent an innovative therapeutic strategy for high-risk cerebral hypoxia-ischemia patients, in particular neonates.

Altered Astrocyte–Neuronal Interactions After Hypoxia-Ischemia in the Neonatal Brain in Female and Male Rats

Background and Purpose— Increased susceptibility to excitotoxicity of the neonatal brain after hypoxia-ischemia (HI) may be caused by limited capacity of astrocytes for glutamate uptake, and mitochondrial failure probably plays a key role in the delayed injury cascade. Male infants have poorer outcome than females after HI, possibly linked to differential intermediary metabolism. Methods— [1-13C]glucose and [1,2-13C]acetate were injected at zero, 6, and 48 hours after unilateral HI in 7-day-old rats. Intermediary metabolism was analyzed with magnetic resonance spectroscopy. Results— Mitochondrial metabolism was generally reduced in the ipsilateral hemisphere for ⩽6 hours after HI, whereas contralaterally, it was reduced in neurons but not in astrocytes. Transfer of glutamate from neurons to astrocytes was increased in the contralateral, but not in the ipsilateral hemisphere at 0 hour, and reduced bilaterally at 6 hours after HI. The transfer of glutamine from astrocytes to glutamatergic neurons was unaltered in both hemispheres, whereas the transfer of glutamine to GABAergic neurons was increased ipsilaterally at 0 hour. Anaplerosis (astrocytes) was decreased, whereas partial pyruvate recycling (astrocytes) was increased directly after HI. Male pups had lower astrocytic mitochondrial metabolism than females immediately after HI, whereas that of females was reduced longer and encompassed both neurons and astrocytes. Conclusions— The prolonged depression in mitochondrial metabolism indicates that mitochondria are vulnerable targets in the delayed injury after neonatal HI. The degree of astrocytic malfunction may be a valid indicator of outcome after hypoxic/HI brain injury and may be linked to the differential outcome in males and females.

Manganese-enhanced magnetic resonance imaging of hypoxic–ischemic brain injury in the neonatal rat

Hypoxic-ischemic injury (HI) to the neonatal brain results in delayed neuronal death with accompanying inflammation for days after the initial insult. The aim of this study was to depict delayed neuronal death after HI using Manganese-enhanced MRI (MEMRI) and to evaluate the specificity of MEMRI in detection of cells related to injury by comparison with histology and immunohistochemistry. 7-day-old Wistar rat pups were subjected to HI (occlusion of right carotid artery and 8% O(2) for 75 min). 16 HI (HI+Mn) and 6 sham operated (Sham+Mn) pups were injected with MnCl(2) (100 mM, 40 mg/kg) and 10 HI-pups (HI+Vehicle) received NaCl i.p. 6 h after HI. 3D T(1)-weighted images (FLASH) and 2D T(2)-maps (MSME) were acquired at 7 T 1, 3 and 7 days after HI. Pups were sacrificed after MR-scanning and brain slices were cut and stained for CD68, GFAP, MAP-2, Caspase-3 and Fluorojade B. No increased manganese-enhancement (ME) was detectable in the injured hemisphere on day 1 or 3 when immunohistochemistry showed massive ongoing neuronal death. 7 days after HI, increased ME was seen on T(1)-w images in parts of the injured cortex, hippocampus and thalamus among HI+Mn pups, but not among HI+Vehicle or Sham+Mn pups. Comparison with immunohistochemistry showed delayed neuronal death and inflammation in these areas with late ME. Areas with increased ME corresponded best with areas with high concentrations of activated microglia. Thus, late manganese-enhancement seems to be related to accumulation of manganese in activated microglia in areas of neuronal death rather than depicting neuronal death per se.

Nanoparticle delivery to the brain--By focused ultrasound and self-assembled nanoparticle-stabilized microbubbles.

The blood-brain barrier (BBB) constitutes a significant obstacle for the delivery of drugs into the central nervous system (CNS). Nanoparticles have been able to partly overcome this obstacle and can thus improve drug delivery across the BBB. Furthermore, focused ultrasound in combination with gas filled microbubbles has opened the BBB in a temporospatial manner in animal models, thus facilitating drug delivery across the BBB. In the current study we combine these two approaches in our quest to develop a novel, generic method for drug delivery across the BBB and into the CNS. Nanoparticles were synthesized using the polymer poly(butyl cyanoacrylate) (PBCA), and such nanoparticles have been reported to cross the BBB to some extent. Together with proteins, these nanoparticles self-assemble into microbubbles. Using these novel microbubbles in combination with focused ultrasound, we successfully and safely opened the BBB transiently in healthy rats. Furthermore, we also demonstrated that the nanoparticles could cross the BBB and deliver a model drug into the CNS.

The Pentose Phosphate Pathway and Pyruvate Carboxylation after Neonatal Hypoxic-Ischemic Brain Injury

The neonatal brain is vulnerable to oxidative stress, and the pentose phosphate pathway (PPP) may be of particular importance to limit the injury. Furthermore, in the neonatal brain, neurons depend on de novo synthesis of neurotransmitters via pyruvate carboxylase (PC) in astrocytes to increase neurotransmitter pools. In the adult brain, PPP activity increases in response to various injuries while pyruvate carboxylation is reduced after ischemia. However, little is known about the response of these pathways after neonatal hypoxia-ischemia (HI). To this end, 7-day-old rats were subjected to unilateral carotid artery ligation followed by hypoxia. Animals were injected with [1,2-13C]glucose during the recovery phase and extracts of cerebral hemispheres ipsi- and contralateral to the operation were analyzed using 1H- and 13C-NMR (nuclear magnetic resonance) spectroscopy and high-performance liquid chromatography (HPLC). After HI, glucose levels were increased and there was evidence of mitochondrial hypometabolism in both hemispheres. Moreover, metabolism via PPP was reduced bilaterally. Ipsilateral glucose metabolism via PC was reduced, but PC activity was relatively preserved compared with glucose metabolism via pyruvate dehydrogenase. The observed reduction in PPP activity after HI may contribute to the increased susceptibility of the neonatal brain to oxidative stress.

Doxycycline treatment in a neonatal rat model of hypoxia-ischemia reduces cerebral tissue and white matter injury: a longitudinal magnetic resonance imaging study.

Doxycycline may potentially be a neuroprotective treatment for neonatal hypoxic–ischemic brain injury through its anti‐inflammatory effects. The aim of this study was to examine any long‐term neuroprotection by doxycycline treatment on cerebral gray and white matter. Hypoxic–ischemic brain injury was induced in 7‐day‐old rats. Pups were treated with either doxycycline (HI+doxy) or saline (HI+vehicle) by intraperitoneal injection at 1 h after hypoxia–ischemia (HI). At 6 h after HI, MnCl2 was injected intraperitoneally for later manganese‐enhanced magnetic resonance imaging (MRI). MRI was performed with diffusion‐weighted imaging on day 1 and T1‐weighted imaging and diffusion tensor imaging at 7, 21 and 42 days after HI. Animals were killed after MRI on day 42 and histological examinations of the brains were performed. There was a tendency towards lower lesion volumes on diffusion maps among HI+doxy than HI+vehicle rats at 1 day after HI. Volumetric MRI showed increasing differences between groups with time after HI, with less cyst formation and less cerebral tissue loss among HI+doxy than HI+vehicle pups. HI+doxy pups had less manganese enhancement on day 7 after HI, indicating reduced inflammation. HI+doxy pups had higher fractional anisotropy on diffusion tensor imaging in major white matter tracts in the injured hemisphere than HI+vehicle pups, indicating less injury to white matter and better myelination. Histological examinations supported the MRI results. Lesion size on early MRI was highly correlated with final injury measures. In conclusion, a single dose of doxycycline reduced long‐term cerebral tissue loss and white matter injury after neonatal HI, with an increasing effect of treatment with time after injury.

No improvement of neuronal metabolism in the reperfusion phase with melatonin treatment after hypoxic-ischemic brain injury in the neonatal rat.

Mitochondrial impairment is a key feature underlying neonatal hypoxic‐ischemic (HI) brain injury and melatonin is potentially neuroprotective through its effects on mitochondria. In this study, we have used 1H and 13C NMR spectroscopy after injection of [1‐13C]glucose and [1,2‐13C]acetate to examine neuronal and astrocytic metabolism in the early reperfusion phase after unilateral HI brain injury in 7‐day‐old rat pups, exploring the effects of HI on mitochondrial function and the potential protective effects of melatonin on brain metabolism. One hour after hypoxia‐ischemia, astrocytic metabolism was recovered and glycolysis was normalized, whereas mitochondrial metabolism in neurons was clearly impaired. Pyruvate carboxylation was also lower in both hemispheres after HI. The transfer of glutamate from neurons to astrocytes was higher whereas the transfer of glutamine from astrocytes to neurons was lower 1 h after HI in the contralateral hemisphere. Neuronal metabolism was equally affected in pups treated with melatonin (10 mg/kg) immediately after HI as in vehicle treated pups indicating that the given dose of melatonin was not capable of protecting the neuronal mitochondria in this early phase after HI brain injury. However, any beneficial effects of melatonin might have been masked by modulatory effects of the solvent dimethyl sulfoxide on cerebral metabolism.

Longitudinal diffusion tensor and manganese-enhanced MRI detect delayed cerebral gray and white matter injury after hypoxia–ischemia and hyperoxia

Background:Hypoxia–ischemia (HI) induces delayed inflammation and long-term gray and white matter brain injury that may be altered by hyperoxia.Methods:HI and 2 h of hyperoxia (100% O2) or room air (21% O2) in 7-d-old (P7) rats were studied by magnetic resonance imaging at 7 Tesla during 42 d: apparent diffusion coefficient (ADC) maps on day 1; T1-weighted manganese-enhanced images on day 7; diffusion tensor images on days 21 and 42; and T2 maps at all time points.Results:The long-term brain tissue destruction on T2 maps was more severe in HI+hyperoxia than HI+room air. ADC was lower in HI+hyperoxia vs. HI+room air and sham and was correlated with long-term outcome. Manganese enhancement indicating inflammation was seen in both the groups along with more microglial activation in HI+hyperoxia on day 7. Fractional anisotropy (FA) in corpus callosum was lower and radial diffusivity was higher in HI+hyperoxia than that in HI+room air and sham on day 21. From day 21 to day 42, FA and radial diffusivity in HI+hyperoxia were unchanged, whereas in HI+room air, FA increased and radial diffusivity decreased to values similar to sham.Conclusion:Hyperoxia caused a more severe tissue destruction, delayed irreversible white matter injury, and increased inflammatory response resulting in a worsening in the trajectory of injury after HI in developing gray and white matter.

Longitudinal Manganese-Enhanced Magnetic Resonance Imaging of Delayed Brain Damage after Hypoxic-Ischemic Injury in the Neonatal Rat

Background: Hypoxia-ischemia (HI) in the neonatal brain results in a prolonged injury process. Longitudinal studies using noninvasive methods can help elucidate the mechanisms behind this process. We have recently demonstrated that manganese-enhanced magnetic resonance imaging (MRI) can depict areas with activated microglia and astrogliosis 7 days after hypoxic-ischemic brain injury. Objective: The current study aimed to follow brain injury after HI in rats longitudinally and compare manganese enhancement of brain areas to the development of injury and presence of reactive astrocytes and microglia. Methods: The Vannucci model for hypoxic-ischemic injury in the neonatal rat was used. Pups were injected with either MnCl2 or saline after 6 h and again on day 41 after HI. Longitudinal MRI (T1 weighted) was performed 1, 3, 7 and 42 days after HI. The brains were prepared for immunohistochemistry after the final MRI. Results: There was severe loss of cerebral tissue from day 7 to day 42 after HI. Most manganese-enhanced areas in the hippocampus, thalamus and basal ganglia at day 7 were liquefied after 42 days. Manganese-enhancement on day 42 corresponded to areas of activated microglia and reactive astrocytes in the remaining cortex, hippocampus and amygdala. However, the main area of enhancement was in the remaining thalamus in a calcified area surrounded by activated microglia and reactive astrocytes. Conclusion: Manganese-enhanced MRI can be a useful tool for in vivo identification of cerebral tissue undergoing delayed cell death and liquefaction after HI. Manganese enhancement at a late stage seems to be related to the accumulation of manganese in calcifications and gliotic tissue.

Diffusion‐weighted MRI for early detection and characterization of prostate cancer in the transgenic adenocarcinoma of the mouse prostate model

To improve early diagnosis of prostate cancer to aid clinical decision‐making. Diffusion‐weighted magnetic resonance imaging (DW‐MRI) is sensitive to water diffusion throughout tissues, which correlates with Gleason score, a histological measure of prostate cancer aggressiveness. In this study the ability of DW‐MRI to detect prostate cancer onset and development was evaluated in transgenic adenocarcinoma of the mouse prostate (TRAMP) mice.