Julie A. Wixey

Post-insult minocycline treatment attenuates hypoxia-ischemia-induced neuroinflammation and white matter injury in the neonatal rat: a comparison of two different dose regimens

An increase in the number of activated microglia in the brain is a key feature of neuroinflammation after a hypoxic–ischemic insult to the preterm neonate and can contribute to white matter injury in the brain. Minocycline is a potent inhibitor of microglia and may have a role as a neuroprotective agent that ameliorates brain injury after hypoxia–ischemia in neonatal animal models. However to date large doses, pre‐insult administration and short periods of treatment after hypoxia–ischemia have mostly been investigated in animal models making it difficult to translate minocyclines potential applicability to protect the human preterm neonatal brain exposed to hypoxia–ischemia. We investigated whether repeated doses of minocycline can minimize white matter injury and neuroinflammation one week after hypoxia–ischemia (right carotid artery ligation and 30 min 6% O2) in the post‐natal day 3 rat pup. Two dosage regimens of minocycline were administered for one week; a high dose of 45 mg/kg 2 h after hypoxia–ischemia then 22.5 mg/kg daily or a low dose 22.5 mg/kg 2 h after hypoxia–ischemia then 10 mg/kg. Post‐natal day 3 hypoxia–ischemia significantly reduced myelin content, numbers of O1‐ and O4‐positive oligodendrocyte progenitor cells and increased activated microglia one week later on post‐natal day 10. The low dose minocycline regimen was as effective as the high dose in ameliorating neuroinflammation after post‐natal day 3 hypoxia–ischemia. However only the high dose regimen significantly attenuated reductions in O1‐ and O4‐positive oligodendrocyte progenitor cells and myelin content. The low dose only significantly attenuated the reduction in O1‐positive oligodendrocyte cell counts. Repeated, daily, post‐insult treatment with minocycline abolished neuroinflammation and may provide neuroprotection to white matter for up to one week after hypoxia–ischemia in a rodent preterm model. The present findings suggest the potential clinical relevance of a repeated, daily minocycline treatment strategy, administered after a hypoxia–ischemia insult, as a therapeutic intervention for hypoxia–ischemia‐affected preterm neonates.

Increased expression of neuronal glucose transporter 3 but not glial glucose transporter 1 following severe diffuse traumatic brain injury in rats.

Traumatic brain injury results in an increased brain energy demand that is associated with profound changes in brain glycolysis and energy metabolism. Increased glycolysis must be met by increasing glucose supply that, in brain, is primarily mediated by two members of the facilitative glucose transporter family, Glut1 and Glut3. Glut1 is expressed in endothelial cells of the blood-brain barrier (BBB) and also in glia, while Glut3 is the primary glucose transporter expressed in neurons. However, few studies have investigated the changes in glucose transporter expression following traumatic brain injury, and in particular, the neuronal and glial glucose transporter responses to injury. This study has therefore focussed on investigating the expression of the glial specific 45-kDa isoform of Glut1 and neuronal specific Glut3 following severe diffuse traumatic brain injury in rats. Following impact-acceleration injury, Glut3 expression was found to increase by at least 300% as early as 4 h after induction of injury and remained elevated for at least 48 h postinjury. The increase in Glut3 expression was clearly evident in both the cerebral cortex and cerebellum. In contrast, expression of the glial specific 45-kDa isoform of Glut1 did not significantly change in either the cerebral cortex or cerebellum following traumatic injury. We conclude that increased glucose uptake after traumatic brain injury is primarily accounted for by increased neuronal Glut 3 glucose transporter expression and that this increased expression after trauma is part of a neuronal stress response that may be involved in increasing neuronal glycolysis and associated energy metabolism to fuel repair processes.

Minocycline: a neuroprotective agent for hypoxic-ischemic brain injury in the neonate?

Minocycline is a second‐generation tetracycline and a potential neuroprotective intervention following brain injury. However, despite the recognized beneficial effects of minocycline in a multitude of adult disease states, the clinical application of minocycline in neonates is contentious. Tetracyclines, as a class, are not usually administered to neonates, but there is compelling evidence that minocycline reduces brain injury after neonatal hypoxic‐ischemic brain injury. This Review focuses on the evidence for minocycline use in neonates by considering aspects of pharmacology, drug regimens, functional outcomes, and mechanisms of action.

Analysis of the CTLA4 Gene in Swedish Coeliac Disease Patients

Background: A genetic susceptibility to coeliac disease is well established, involving HLA and nonHLA components. CTLA4 is an important regulator of T-cell function and some studies have suggested that sequence variation in the gene might be a determinant of disease susceptibility, although the evidence is conflicting. Methods: Sixty-two children with biopsy-proven coeliac disease attending a single centre in Sweden were studied. All were genotyped for presence of the HLA-DQA1*0501, B1*0201 alleles. Those who carried the HLA-DQ heterodimer (58/62) were genotyped for the +49 (A/G) exon 1 polymorphism. The transmission disequilibrium test (TDT) was used to test for association between coeliac disease and the A allele. The entire CTLA4 gene was screened for other sequence variants using a combination of conformation-sensitive gel electrophoresis and direct sequencing. Results: A significant association between the exon 1 polymorphism and coeliac disease was observed ( P = 0.02). No other sequence variants in CTLA4 were detected. Conclusions: This study provides further evidence that variation in CTLA4 is a determinant of coeliac disease susceptibility. If not mediated through the +49 (A/G) dimorphism directly, then the effect is likely to be mediated through linkage disequilibrium.

Selective Losses of Brainstem Catecholamine Neurons After Hypoxia-Ischemia in the Immature Rat Pup

Hypoxic-ischemic (HI) injury in the preterm neonate incurs numerous functional deficits, however little is known about the neurochemically-defined brain nuclei that may underpin them. Key candidates are the brainstem catecholamine neurons. Using an immature animal model, the postnatal day (P)-3 (P3) rat pup, we investigated the effects of HI on brainstem catecholamine neurons in the locus coeruleus, nucleus tractus solitarius (NTS), and ventrolateral medulla (VLM). On P21, we found that prior P3 HI significantly reduced numbers of catecholaminergic neurons in the locus coeruleus, NTS, and VLM. Only locus coeruleus A6, NTS A2, and VLM A1 noradrenergic neurons, but not NTS C2 and VLM C1 adrenergic neurons, were lost. There was also an associated reduction in dopamine-beta-hydroxylase-positive immunolabeling in the forebrain. These findings suggest neonatal HI can affect specific neurochemically-defined neuronal populations in the brainstem and that noradrenergic neurons are particularly vulnerable to HI injury.

Delayed P2X4R expression after hypoxia-ischemia is associated with microglia in the immature rat brain.

In a preterm hypoxia-ischemia model in the post-natal day 3 rat, we characterized how the expression of purine ionotropic P2X(4) receptors change in the brain post-insult. After hypoxia-ischemia, P2X(4) receptor expression increased significantly and was associated with a late increase in ionised calcium binding adapter molecule-1 protein expression indicative of microglia cell activation. Minocycline, a potent inhibitor of microglia, attenuated the hypoxia-ischemia-induced increase in P2X(4) receptor expression. We postulate that P2X(4) receptor-positive microglia may represent a population of secondary injury-induced activated microglia. Future studies will determine whether this population contributes to the progression of injury in the immature brain.

Efficacy of post-insult minocycline administration to alter long-term hypoxia-ischemia-induced damage to the serotonergic system in the immature rat brain

Neuroinflammation is a key mechanism contributing to long-term neuropathology observed after neonatal hypoxia-ischemia (HI). Minocycline, a second-generation tetracycline, is a potent inhibitor of neuroinflammatory mediators and is successful for at least short-term amelioration of neuronal injury after neonatal HI. However the long-term efficacy of minocycline to prevent injury to a specific neuronal network, such as the serotonergic (5-hydroxytryptamine, 5-HT) system, is not known. In a post-natal day 3 (P3) rat model of preterm HI we found significant reductions in 5-HT levels, 5-HT transporter expression and numbers of 5-HT-positive dorsal raphé neurons 6 weeks after insult compared to control animals. Numbers of activated microglia were significantly elevated in the thalamus and dorsal raphé although the greatest numbers were observed in the thalamus. Brain levels of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) were also significantly elevated on P45 in the thalamus and frontal cortex. Post-insult administration of minocycline for 1 week (P3-P9) attenuated the P3 HI-induced increases in numbers of activated microglia and levels of TNF-α and IL-1β on P45 with concurrent changes in serotonergic outcomes. The parallel prevention of P3 HI-induced serotonergic changes suggests that inhibition of neuroinflammation within the first week after P3 HI injury was sufficient to prevent long-term neuroinflammation as well as serotonergic system damage still evident at 6 weeks. Thus early, post-insult administration of minocycline may target secondary neuroinflammation and represent a long-term therapy to preserve the integrity of the central serotonergic network in the preterm neonate.

Ibuprofen inhibits neuroinflammation and attenuates white matter damage following hypoxia–ischemia in the immature rodent brain

Damage to major white matter tracts is a hallmark mark feature of hypoxic-ischemic (HI) brain injury in the preterm neonate. There is, however, no therapeutic intervention to treat this injury. Neuroinflammation is thought to play a prominent role in the pathogenesis of the HI-induced white matter damage but identification of the key mediators that constitute the inflammatory response remain to be fully elucidated. Cyclooxygenase enzymes (COX-1 and COX-2) are candidate neuroinflammatory mediators that may contribute to the HI-induced demise of early oligodendrocyte progenitors and myelination. We investigated whether ibuprofen, a non-steroidal anti-inflammatory drug that inhibits COX enzymes, can attenuate neuroinflammation and associated white matter damage incurred in a rodent model of preterm HI. On postnatal day 3 (P3), HI was produced (right carotid artery ligation and 30 min 6% O(2)). An initial dose of ibuprofen (100mg/kg, s.c.) was administered 2h after HI followed by a maintenance dose (50mg/kg, s.c.) every 24h for 6 days. Post-HI ibuprofen treatment significantly attenuated the P3 HI-induced increases in COX-2 protein expression as well as interleukin-1beta (IL-1β) and tumour necrosis factor-alpha (TNF-α) levels in the brain. Ibuprofen treatment also prevented the HI-induced loss O4- and O1-positive oligodendrocyte progenitor cells and myelin basic protein (MBP)-positive myelin content one week after P3 HI. These findings suggest that a repeated, daily, ibuprofen treatment regimen administered after an HI insult may be a potential therapeutic intervention to prevent HI-induced damage to white matter progenitors and early myelination in the preterm neonate.

Inhibition of neuroinflammation prevents injury to the serotonergic network after hypoxia-ischemia in the immature rat brain

The phenotypic identities and characterization of neural networks disrupted after neonatal hypoxia-ischemia (HI) in the preterm brain remain to be elucidated. Interruption of the central serotonergic (5-hydroxytryptamine [5-HT]) system can lead to numerous functional deficits, many of which match those in human preterm neonates exposedto HI. How the central serotonergic network is damaged after HIand mechanisms underlying such injury are not known. We used aPostnatal Day 3 rat model of preterm HI and found parallel reductionsin the 5-HT transporter expression, 5-HT levels and numbers of 5-HT-positive dorsal raphe neurons 1 week after insult. Post-HI administration of minocycline, an inhibitor of activated microglia, attenuated HI-induced damage to the serotonergic network. Minocycline effects seemed to be region specific, that is, where there was microglialactivation and increases in tumor necrosis factor-&agr; and interleukin1&bgr;. The concurrent improvement in serotonergic outcomes suggests that inhibition of neuroinflammation prevented damage to theserotonergic neurons rather than affected the regulation of 5-HT orserotonin transporter. These data elucidate the mechanisms of serotonergic network injury in HI, and despite the known adverse effects associated with the use of minocycline in neonates, postinsult administration of minocycline may represent a novel approach to counter neuroinflammation and preserve the integrity of the central serotonergic network in the preterm neonate.

Long-term losses of amygdala corticotropin-releasing factor neurons are associated with behavioural outcomes following neonatal hypoxia-ischemia

Neuronal losses are observed in the brain after neonatal hypoxia-ischemia (HI) however few studies have examined the effects of HI on specific neuronal phenotypes and their possible contribution to behavioural outcomes. In the present study we examined whether postnatal day 3 (P3) HI alters numbers of corticotropin-releasing factor (CRF) and neuropeptide-Y (NPY) neurons in the paraventricular nucleus of the hypothalamus (PVN), the bed nucleus of the stria terminalis (BNST) and the amygdala, 1 (P10) and 6 (P45) weeks after P3 HI. A significant reduction in the number of CRF-positive neurons in the PVN, central nucleus of the amygdala (CeA) and BNST ipsilateral to the carotid ligation 1 and 6 weeks after P3 HI was observed. There was also a significant reduction in the number of NPY-positive neurons in the PVN, amygdala and BNST ipsilateral to the carotid ligation 1 week after P3 HI. However after 6 weeks, only the number of PVN NPY-positive neurons decreased significantly. At 6 weeks post-insult, the number of CeA CRF-positive neurons was inversely associated with locomotor activity and exploratory behaviour in an open field. In contrast, no significant correlations between neuronal counts and early neurodevelopment tests performed on P10 were observed. Thus after P3 HI persistent losses of CRF- and NPY-positive neurons occur and the loss of CeA CRF neurons may provide a central anatomical mechanism underlying neurobehavioural deficits observed 6 weeks after P3 HI.