minocycline and Hypoxia-Ischemia--Brain

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.

Topics: Animals; Animals, Newborn; Anti-Inflammatory Agents; Antioxidants; Apoptosis; Blood-Brain Barrier; Brain; Humans; Hypoxia-Ischemia, Brain; Infant Behavior; Infant, Newborn; Matrix Metalloproteinase Inhibitors; Minocycline; Necrosis; Neurons; Neuroprotective Agents

Minocycline is a widely used tetracycline antibiotic. For decades, it has been used to treat various gram-positive and gram-negative infections. Minocycline was recently shown to have neuroprotective properties in animal models of acute neurologic injury. As a neuroprotective agent, the drug appears more effective than other treatment options. In addition to its high penetration of the blood-brain barrier, minocycline is a safe compound commonly used to treat chronic infections. Its several mechanisms of action in neuroprotection -- antiinflammatory and antiapoptotic effects, and protease inhibition -- make it a desirable candidate as therapy for acute neurologic injury, such as ischemic stroke. Minocycline is ready for clinical trials of acute neurologic injury.

Topics: Animals; Anti-Bacterial Agents; Anti-Inflammatory Agents; Apoptosis; Brain Injuries; Humans; Hypoxia-Ischemia, Brain; Matrix Metalloproteinases; Minocycline; Neuroprotective Agents; Stroke

Ischemic animal model studies have shown a neuroprotective effect of minocycline.. To analyze the effect of minocycline treatment in human acute ischemic stroke.. We performed an open-label, evaluator-blinded study. Minocycline at a dosage of 200 mg was administered orally for 5 days. The therapeutic window of time was 6 to 24 hours after onset of stroke. Data from NIH Stroke Scale (NIHSS), modified Rankin Scale (mRS), and Barthel Index (BI) were evaluated. The primary objective was to compare changes from baseline to day 90 in NIHSS in the minocycline group vs placebo.. One hundred fifty-two patients were included in the study. Seventy-four patients received minocycline treatment, and 77 received placebo. NIHSS and mRS were significantly lower and BI scores were significantly higher in minocycline-treated patients. This pattern was already apparent on day 7 and day 30 of follow-up. Deaths, myocardial infarctions, recurrent strokes, and hemorrhagic transformations during follow-up did not differ by treatment group.. Patients with acute stroke had significantly better outcome with minocycline treatment compared with placebo. The findings suggest a potential benefit of minocycline in acute ischemic stroke.

Topics: Acute Disease; Aged; Anti-Bacterial Agents; Anti-Inflammatory Agents; Apoptosis; Cerebral Hemorrhage; Female; Gliosis; Humans; Hypoxia-Ischemia, Brain; Male; Microglia; Middle Aged; Minocycline; Mortality; Myocardial Infarction; Neuroprotective Agents; Placebos; Secondary Prevention; Signal Transduction; Single-Blind Method; Stroke; Treatment Outcome

Neonatal hypoxic injury (NHI) is a devastating cause of disease that affects >60% of babies born with a very low birth weight, resulting in significant morbidity and mortality, including life-long neurological consequences such as seizures, cerebral palsy, and intellectual disability. Hypoxic injury results in increased neuronal death, which disrupts normal brain development. Although animal model systems have been useful to study the effects of NHI, they do not fully represent the uniqueness and complexities of the human brain. To better understand the effects of hypoxia on human brain development, we have generated a brain organoid protocol and evaluated these cells over the course of 6 months. As anticipated, the expression of a forebrain marker, FOXG1, increased and then remained expressed over time, while there was a transition in the expression of the deep-layer (TBR1) and upper-layer (SATB2) cortical markers. In addition, ventral genes (Eng1 and Nkx2.1) as well as markers of specialized nonneuronal cells (Olig2 and GFAP) also increased at later time points. We next tested the development of our in vitro cerebral organoid model at different oxygen concentrations and found that hypoxia repressed gene markers for forebrain, oligodendrocytes, glial cells, and cortical layers, as well as genes important for the migration of cortical neurons. In contrast, ventral markers were either unaffected or even increased in expression with hypoxic insult. Interestingly, the negative effect of hypoxia on the dorsal brain genes as well as oligodendrocytes, and neuronal progenitors could be mitigated by the use of minocycline, an FDA-approved small molecule. Taken together, we have generated a unique and relevant in vitro human brain model system to study diseases such as NHI as well as their potential treatments. Using this system, we have shown the efficacy of minocycline for human NHI.

Topics: Brain; Cell Death; Human Embryonic Stem Cells; Humans; Hypoxia-Ischemia, Brain; Hypoxia, Brain; Minocycline; Neurons; Organoids; Proto-Oncogene Proteins c-bcl-2; SOXB1 Transcription Factors; Time Factors

It has become increasingly evident the serotonergic (5-hydroxytryptamine, 5-HT) system is an important central neuronal network disrupted following neonatal hypoxic-ischemic (HI) insults. Serotonin acts via a variety of receptor subtypes that are differentially associated with behavioural and cognitive mechanisms. The 5-HT

Topics: Animals; Brain; Female; Hypoxia-Ischemia, Brain; Ischemia; Microglia; Minocycline; Neurons; Rats, Sprague-Dawley; Receptors, Serotonin; Serotonin

We previously found increased microglial proliferation and pro-inflammatory cytokine release in infant mice compared to juvenile mice after hypoxia-ischemia (HI). The aim of the current study was to assess for differences in the effect of microglial suppression on HI-induced brain injury in infant and juvenile mice. HI was induced in neonatal (P9) and juvenile (P30) mice and minocycline or vehicle was administered at 2h and 24h post-HI. P9 minocycline-treated mice demonstrated early but transient improvements in neurologic injury, while P30 minocycline-treated mice demonstrated sustained improvements in cerebral atrophy and Morris Water Maze performance at 60days post-HI.

Topics: Aging; Animals; Animals, Newborn; Brain; Brain Injuries; CD11b Antigen; Disease Models, Animal; Flow Cytometry; Functional Laterality; Hypoxia-Ischemia, Brain; Learning Disabilities; Leukocyte Common Antigens; Magnetic Resonance Imaging; Maze Learning; Mice; Microglia; Minocycline; Neurologic Examination; Statistics, Nonparametric; Time Factors

The biology of cerebral white matter injury has been woefully understudied, in part because of the difficulty of reliably modeling this type of injury in rodents. Periventricular leukomalacia (PVL) is the predominant form of brain injury and the most common cause of cerebral palsy in premature infants. PVL is characterized by predominant white matter injury. No specific therapy for PVL is presently available, because the pathogenesis is not well understood. Here we report that two types of mouse PVL models have been created by hypoxia-ischemia with or without systemic coadministration of lipopolysaccharide (LPS). LPS coadministration exacerbated hypoxic-ischemic white matter injury and led to enhanced microglial activation and astrogliosis. Drug trials with the antiinflammatory agent minocycline, the antiexcitotoxic agent NBQX, and the antioxidant agent edaravone showed various degrees of protection in the two models, indicating that excitotoxic, oxidative, and inflammatory forms of injury are involved in the pathogenesis of injury to immature white matter. We then applied immunoelectron microscopy to reveal fine structural changes in the injured white matter and found that synapses between axons and oligodendroglial precursor cells (OPCs) are quickly and profoundly damaged. Hypoxia-ischemia caused a drastic decrease in the number of postsynaptic densities associated with the glutamatergic axon-OPC synapses defined by the expression of vesicular glutamate transporters, vGluT1 and vGluT2, on axon terminals that formed contacts with OPCs in the periventricular white matter, resulted in selective shrinkage of the postsynaptic OPCs contacted by vGluT2 labeled synapses, and led to excitotoxicity mediated by GluR2-lacking, Ca(2+) -permeable AMPA receptors. Overall, the present study provides novel mechanistic insights into the pathogenesis of PVL and reveals that axon-glia synapses are highly vulnerable to white matter injury in the developing brain. More broadly, the study of white matter development and injury has general implications for a variety of neurological diseases, including PVL, stroke, spinal cord injury, and multiple sclerosis.

Topics: Animals; Animals, Newborn; Antigens; Brain Injuries; Carotid Artery Diseases; Disease Models, Animal; Excitatory Amino Acid Antagonists; Functional Laterality; Glial Fibrillary Acidic Protein; Hypoxia-Ischemia, Brain; Leukoencephalopathies; Luminescent Proteins; Mice; Mice, Inbred C57BL; Mice, Transgenic; Microscopy, Electron, Transmission; Minocycline; Myelin Basic Protein; Nerve Fibers, Myelinated; Neuroglia; Polysaccharides; Proteoglycans; Quinoxalines; Receptors, AMPA; Synapses; Vesicular Glutamate Transport Protein 1; Vesicular Glutamate Transport Protein 2

Neuronal injury is a key feature of neonatal hypoxic-ischemic (HI) brain injury. However, the mechanisms underpinning neuronal losses, such as in the brainstem, are poorly understood. One possibility is that disrupted neural connections between the cortex and brainstem may compromise the survival of neuronal cell bodies in the brainstem. We investigated whether brainstem raphé serotonergic neurons that project to the cortex are lost after HI. We also tested if neuroinflammation has a role in disrupting brainstem raphé projections. Postnatal day 3 (P3) rats underwent unilateral carotid artery ligation followed by hypoxia (6% oxygen for 30 min). A retrograde tracer, choleratoxin b, was deposited in the motor cortex on P38. On P45 we found that retrogradely labelled neurons in the dorsal raphé dorsal, ventrolateral, interfascicular, caudal and ventral nuclei were lost after P3 HI. All retrogradely labelled neurons in the raphé nuclei were serotonergic. Numbers of retrogradely labelled neurons were also reduced in the ventromedial thalamus and basolateral amygdala. Minocycline treatment (45 mg/kg 2 h post-HI, 22.5 mg/kg daily P4-P9) attenuated losses of retrogradely labelled neurons in the dorsal raphé ventrolateral, interfascicular and ventral raphé nuclei, and the ventromedial thalamus. These results indicate that raphé neurons projecting to the cortex constitute a population of serotonergic neurons that are lost after P3 HI. Furthermore, neuroinflammation has a role in the disruption of raphé and thalamic neural projections. Future studies investigating the cellular mechanisms of axonal degeneration may reveal new targets for interventions to prevent neuronal losses after neonatal HI.

Topics: Amygdala; Animals; Anti-Bacterial Agents; Carotid Arteries; Cholera Toxin; Hypoxia-Ischemia, Brain; Inflammation; Minocycline; Motor Cortex; Neural Pathways; Neuronal Tract-Tracers; Raphe Nuclei; Rats; Rats, Sprague-Dawley; Serotonergic Neurons; Thalamus

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 exposed to HI. How the central serotonergic network is damaged after HI and mechanisms underlying such injury are not known. We used a Postnatal Day 3 rat model of preterm HI and found parallel reductions in 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 micro-glial activation and increases in tumor necrosis factor-α and inter-leukin 1β. The concurrent improvement in serotonergic outcomes suggests that inhibition of neuroinflammation prevented damage to the serotonergic neurons rather than affected the regulation of 5-HT or serotonin 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.

Topics: Animals; Animals, Newborn; Anti-Inflammatory Agents, Non-Steroidal; Female; Hypoxia-Ischemia, Brain; Inflammation; Inflammation Mediators; Minocycline; Nerve Net; Prosencephalon; Random Allocation; Rats; Rats, Sprague-Dawley; RNA-Binding Proteins; Serotonin

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.

Topics: Age Factors; Animals; Animals, Newborn; Encephalitis; Female; Hypoxia-Ischemia, Brain; Male; Minocycline; Nerve Degeneration; Neuroprotective Agents; Raphe Nuclei; Rats; Rats, Sprague-Dawley; Serotonin

To establish a model of immature rat hypoxic-ischemic brain damage (HIBD) which was expected to be similar to periventricular leukomalacia in human preterm infants pathologically and neuroethologically, and to investigate the role of minocycline (MN) in this model.. Totally 192 Sprague-Dawley rats (postnatal day 2, P(2)), of either sex, were randomly divided into 4 groups: normal-group, sham operation group, HIBD-group, HIBD + MN group, each group had 48 rats. HIBD group and HIBD + MN group survived the left common carotid artery (CCA) ligation followed by 4h exposure to 8% O(2). Rats in sham operation group only survived the left CCA isolation. Rats in normal group were not treated with anything. In HIBD + MN group, the rats were treated with intraperitoneal injection of minocycline 45 mg/kg, immediately after HI and every 24 h for 2 days. Brain tissues were collected on day 3, 1 week, 2 weeks, 4 weeks after HI, for hematoxylin-eosin staining and histological scoring. Frozen sections of the brains were stained with anti-O4, anti-O1 immunohistochemistry on day 3 after HI, and MBP immunohistochemistry 2 weeks after HI. Rats in the four groups underwent neuroethologic examination 4 weeks after HI.. In the HIBD group, there were pathological changes in the periventricular white matter. The pathological changes were milder in HIBD + MN group; There was no statistically significant difference between the normal group and HIBD + MN group in the number of positively stained O4 cell (P > 0.05). The number of positively stained O4 cell in the HIBD group was significantly reduced, compared with that of normal group, sham operation group, and HIBD + MN group (23.67 ± 12.00 vs. 52.89 ± 10.68, 39.28 ± 11.78, 41.63 ± 8.41, P < 0.05). The differences in the number of positively stained O1 cell among the normal group, sham operation group, HIBD group and HIBD + MN group had no statistical significance (P = 0.093). The numbers of myelin basic protein (MBP) positively immunostained fiber bundles in the HIBD + MN group were significantly less than that of the normal group and sham operation group (P < 0.05). The numbers of MBP positively immunostained fiber bundles in the HIBD group were significantly less than that of the normal group, sham operation group, and HIBD + MN group (14.71 ± 7.42 vs. 36.67 ± 6.50, 35.50 ± 3.24, 26.33 ± 5.92, P < 0.05). The HIBD group had long-term neuroethologic abnormality. There was no statistically significant difference in the inclined plane test, hanging test and cylinder test among the HIBD + MN group, normal group, and sham operation group (P > 0.05). The scores of the HIBD group had statistical significantly among the normal group, sham operation group and HIBD + MN group (P < 0.05). In the open field test, there was no statistically significant difference between the HIBD group and HIBD + MN group (P = 0.772), but there was significant difference between these two groups and the normal group, sham operation group (P < 0.05).. Minocycline protects the pre-oligodendrocyte and has protective effects in terms of long-term neuroethology.

Topics: Animals; Animals, Newborn; Disease Models, Animal; Hypoxia-Ischemia, Brain; Minocycline; Rats; Rats, Sprague-Dawley

Perinatal hypoxia-ischemia (H-I) often manifests as cognitive and/or motor disturbances that appear early in development. Growing evidence indicates that neuroinflammation may exacerbate H-I injury. Resident microglia release proinflammatory cytokines and proteases in response to ischemia. Matrix metalloproteinases (MMPs), in particular, activate cytokines and degrade basement membrane proteins. These actions ultimately permit entry of peripheral leukocytes into the CNS neuropil, enhancing neuroinflammation and cell death. Currently, the relative contributions of resident and peripheral immune cells to ischemic brain injury are unclear. The present study employed an ex vivo model of H-I through oxygen glucose deprivation (OGD) to identify the cellular localization of MMP-9 in organotypic hippocampal slices from rat, and to determine whether inhibiting gelatin-degrading MMPs affords neuroprotection in the absence of peripheral immune cells. Immunohistochemistry revealed ubiquitous neuronal MMP-9 expression in both normoxic and hypoxic slices. Increased MMP-9 expression was detected in CD11b-positive microglia after 48 h exposure to OGD relative to normoxic controls. Consistent with these data, in situ zymography showed increased gelatinolytic activity after OGD. Gelatin-cleaved fluorescence localized to astrocytic processes and somata of various cellular morphologies. Treatment with either the MMP inhibitor AG3340 (prinomastat) or minocycline dampened OGD-induced gelatinolytic activity and neural injury, as measured by Fluoro-Jade staining, relative to vehicle controls. These results show that resident microglia, in the absence of peripheral immune cells, were sufficient to enhance neural injury after OGD in the organotypic hippocampal slice. Additionally, these effects were associated with upregulation or secretion of MMP-9, and were blocked after treatment with either the gelatinase-selective compound AG3340 or the anti-inflammatory compound minocycline. These data, coupled with the effectiveness of these compounds previously shown in vivo, support the selective targeting of gelatin-degrading MMPs and activated microglia as potential therapeutic approaches to combat neonatal H-I injury.

Topics: Animals; Animals, Newborn; Anti-Inflammatory Agents; Astrocytes; Brain Infarction; Enzyme Inhibitors; Gliosis; Hypoxia-Ischemia, Brain; Immunohistochemistry; Matrix Metalloproteinase 9; Matrix Metalloproteinase Inhibitors; Microglia; Minocycline; Nerve Degeneration; Neurons; Organ Culture Techniques; Organic Chemicals; Rats; Rats, Sprague-Dawley; Up-Regulation

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.

Topics: Animals; Brain Chemistry; Calcium-Binding Proteins; Corpus Callosum; Female; Hypoxia-Ischemia, Brain; Immunohistochemistry; Male; Microfilament Proteins; Microglia; Minocycline; Rats; Rats, Sprague-Dawley; Receptors, Purinergic P2; Receptors, Purinergic P2X4

Hypoxia-ischemia (H-I) can produce widespread neurodegeneration and deep cerebral white matter injury in the neonate. Resident microglia and invading leukocytes promote lesion progression by releasing reactive oxygen species, proteases and other pro-inflammatory mediators. After injury, expression of the gelatin-degrading matrix metalloproteinases (MMPs), MMP-2 and MMP-9, are thought to result in the proteolysis of extracellular matrix (ECM), activation of cytokines/chemokines, and the loss of vascular integrity. Thus, therapies targeting ECM degradation and progressive neuroinflammation may be beneficial in reducing H-I - induced neuropathy. Minocycline has MMP-inhibitory properties and is both anti-inflammatory and neuroprotective. AG3340 (prinomastat) is an MMP inhibitor with high selectivity for the gelatinases. The purpose of this study was to determine whether these compounds could limit H-I--induced injury when administered at a delayed time point.. Sprague-Dawley rats were exposed to H-I at postnatal day 7 (P7), consisting of unilateral carotid artery ligation followed by 90 min exposure to 8% O2. Minocycline, AG3340, or vehicle were administered once daily for 6 days, beginning 24 hours after insult. Animals were sacrificed at P14 for neurohistological assessments. Immunohistochemistry was performed to determine the degree of reactive astrogliosis and immune cell activation/recruitment. Neural injury was detected using the Fluoro-Jade stain, a marker that identifies degenerating cells.. CD11b and glial fibrillary acidic protein (GFAP) immunopositive cells increased in ipsilateral cortex after treatment with vehicle alone, demonstrating microglia/macrophage recruitment and reactive astrogliosis, respectively. Fluoro-Jade staining was markedly increased throughout the fronto-parietal cortex, striatum and hippocampus. Treatment with minocycline or AG3340 inhibited microglia/macrophage recruitment, attenuated astrogliosis and reduced Fluoro-Jade staining when compared to vehicle alone.. The selective gelatinase inhibitor AG3340 showed equal efficacy in reducing neural injury and dampening neuroinflammation when compared to the anti-inflammatory compound minocycline. Thus, MMP-2 and MMP-9 may be viable therapeutic targets to treat neonatal brain injury.

Topics: Animals; Animals, Newborn; Brain Injuries; Enzyme Inhibitors; Hypoxia-Ischemia, Brain; Macrophages; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Matrix Metalloproteinase Inhibitors; Microglia; Minocycline; Neuroprotective Agents; Organic Chemicals; Random Allocation; Rats; Rats, Sprague-Dawley

Periventricular white matter injury in premature infants occurs following hypoxia/ischaemia and systemic infection, and results in hypomyelination, as well as neuromotor and cognitive deficits later in life. Inflammatory infiltrates are seen within human cerebral white matter from periventricular leucomalacia (PVL) cases.. In this study, we examine the time course of CD-68+ microglial cell responses relative to cell death within white matter following hypoxia/ischaemia in a rat model of PVL. We also tested the efficacy of the minocycline, an agent that suppresses microglial activation, in this model when administered as a post-insult treatment.. We show that preoligodendrocyte injury in the post-natal day 6 begins within 24 h and continues for 48-96 h after hypoxia/ischaemia, and that microglial responses occur primarily over the first 96 h following hypoxia/ischaemia. Minocycline treatment over this 96 h time window following the insult resulted in significant protection against white matter injury, and this effect was concomitant with a reduction in CD-68+ microglial cell numbers.. These results suggest that anti-inflammatory treatments may represent a useful strategy in the treatment of PVL, where clinical conditions would favour a post-insult treatment strategy.

Topics: Animals; Animals, Newborn; Cell Death; Disease Models, Animal; Humans; Hypoxia-Ischemia, Brain; Hypoxia, Brain; Infant, Newborn; Leukomalacia, Periventricular; Microglia; Minocycline; Myelin Basic Protein; Rats; Rats, Long-Evans; Tegmentum Mesencephali

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 minocycline's 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% O(2)) 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 2h after hypoxia-ischemia then 22.5 mg/kg daily or a low dose 22.5 mg/kg 2h 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.

Topics: Animals; Animals, Newborn; Anti-Bacterial Agents; Brain Injuries; Disease Models, Animal; Dose-Response Relationship, Drug; Encephalitis; Hypoxia-Ischemia, Brain; Injections, Intraperitoneal; Microglia; Minocycline; Myelin Sheath; Nerve Tissue; Neuroprotective Agents; Oligodendroglia; Rats; Rats, Sprague-Dawley; Stem Cells; Time Factors

Hypoxic-ischemic (HI) brain injury in the perinatal period causes significant morbidity. Minocycline (MN) is a tetracycline derivative that has reduced brain injury in various animal models of neurodegeneration, including perinatal ischemia. To determine whether MN can modulate the expression of chemokine receptors and interleukin-10 (IL10) in a model of neonatal brain injury, we produced an HI insult to the right cerebral hemisphere (ipsilateral) of the 7-day-old rat (PD7) by right common carotid artery ligation and 2.25 hr of hypoxia in 8% oxygen. MN (45 mg/kg, i.p.) or vehicle (PBS) was injected twice: 2 days and immediately before the HI insult. At 0, 1, 3, and 24 hr and 14 days after HI, total RNA from the ipsilateral and contralateral (exposed to hypoxia only) hemispheres was extracted, reverse transcribed, and amplified with gene-specific primers using a semiquantitative RT-PCR for macrophage inflammatory protein-1alpha), interferon-inducible protein (IP-10), C-C chemokine receptor 5 (CCR5; MIP-1alpha receptor), C-X-C chemokine receptor 3 (CXCR3; IP-10 receptor), and IL10. We found that, in the ipsilateral hemisphere, a significant (P < 0.05) increase in MIP-1alpha, IP-10, CCR5, and CXCR3 mRNA levels was observed. MN treatment decreased mRNA levels for CCR5 and CXCR3. In contrast, the levels of antiinflammatory cytokine IL10 were markedly decreased as a result of HI insult. Treatment with MN, however, had no effect on IL10. We conclude that MN decreased proinflammatory chemokine receptor expression but had little or no influence on the expression of antiinflammatory cytokine IL10. These effects confirm the antiinflammatory effect of MN in neonatal HI brain injury.

Topics: Animals; Animals, Newborn; Blotting, Western; Functional Laterality; Hypoxia-Ischemia, Brain; Interleukin-10; Minocycline; Neuroprotective Agents; Rats; Receptors, Chemokine; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

The role of minocycline in preventing white matter injury, in particular the injury to developing oligodendrocytes was examined in a neonatal rat model of hypoxia-ischemia. Hypoxia-ischemia was achieved through bilateral carotid artery occlusion followed by exposure to hypoxia (8% oxygen) for 15 min in postnatal day 4 Sprague-Dawley rats. A sham operation was performed in control rats. Minocycline (45 mg/kg) or normal phosphate-buffered saline was administered intraperitoneally 12 h before and immediately after bilateral carotid artery occlusion+hypoxia and then every 24 h for 3 days. Nissl staining revealed pyknotic cells in the white matter area of the rat brain 1 and 5 days after hypoxia-ischemia. Hypoxia-ischemia insult also resulted in apoptotic oligodendrocyte cell death, loss of O4+ and O1+ oligodendrocyte immunoreactivity, and hypomyelination as indicated by decreased myelin basic protein immunostaining and by loss of mature oligodendrocytes in the rat brain. Minocycline significantly attenuated hypoxia-ischemia-induced brain injury. The protective effect of minocycline was associated with suppression of hypoxia-ischemia-induced microglial activation as indicated by the decreased number of activated microglia, which were also interleukin-1beta and inducible nitric oxide synthase expressing cells. The protective effect of minocycline was also linked with reduction in hypoxia-ischemia-induced oxidative and nitrosative stress as indicated by 4-hydroxynonenal and nitrotyrosine positive oligodendrocytes, respectively. The reduction in hypoxia-ischemia-induced oxidative stress was also evidenced by the decreases in the content of 8-isoprostane in the minocycline-treated hypoxia-ischemia rat brain as compared with that in the vehicle-treated hypoxia-ischemia rat brain. The overall results suggest that reduction in microglial activation may protect developing oligodendrocytes in the neonatal brain from hypoxia-ischemia injury.

Topics: Animals; Animals, Newborn; Antigens, Surface; Biomarkers; Brain; Carotid Artery, Common; Cell Differentiation; Disease Models, Animal; Free Radicals; Gliosis; Hypoxia-Ischemia, Brain; Ligation; Microglia; Minocycline; Nerve Degeneration; Nerve Fibers, Myelinated; Nerve Regeneration; Neuroprotective Agents; Oligodendroglia; Oxidative Stress; Rats; Rats, Sprague-Dawley; Stem Cells; Treatment Outcome

The tetracycline derivatives minocycline (MINO) and doxycycline (DOXY) have been shown to be neuroprotective in in vivo and in vitro models of stroke. This neuroprotection is thought to be due to the suppression of microglial activation. However, the specific molecular parameters in microglia of the tetracyclines' effect are not understood. We subjected cultured rat microglial and neuronal cells to in vitro hypoxia and examined the effects of MINO and DOXY pre-treatments. Our data showed that MINO and DOXY protect against hypoxia-induced neuronal death by a mechanism dependent on regulation of microglial factors, but likely unrelated to regulation of microglial proliferation/viability. Both MINO and DOXY suppressed the hypoxic activation of ED-1, a marker for microglial activation. Morphological analyses of hypoxic microglia using the microglial marker Iba1 revealed that treatment with MINO and DOXY caused a higher percentage of microglia to remain in a non-activated state. MINO suppressed the hypoxic upregulation of pro-inflammatory agents nitric oxide (NO), interleukin-1 beta (IL-1beta), and tumor necrosis factor alpha (TNF-alpha), while DOXY down-regulated only NO and IL-1beta. In contrast, the hypoxic activation of pro-survival/neuroprotective microglial proteins, such as brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF), were unaffected by tetracycline treatments. Taken together, these results suggest that MINO and DOXY may provide neuroprotection against stroke by selectively down-regulating microglial toxic factors while maintaining functional pro-survival factors.

Topics: Animals; Animals, Newborn; Biomarkers; Brain; Cell Proliferation; Cell Survival; Cells, Cultured; Coculture Techniques; Doxycycline; Ectodysplasins; Encephalitis; Hypoxia-Ischemia, Brain; Inflammation Mediators; Membrane Proteins; Microglia; Minocycline; Nerve Growth Factors; Neuroprotective Agents; Rats; Rats, Sprague-Dawley; Tetracyclines; Tumor Necrosis Factors; Up-Regulation

To investigate whether minocycline provides long-lasting protection against neonatal hypoxia-ischemia-induced brain injury and neurobehavioral deficits, minocycline was administered intraperitoneally in postnatal day 4 Sprague-Dawley rats subjected to bilateral carotid artery occlusion followed by exposure to hypoxia (8% oxygen for 15 min). Brain injury and myelination were examined on postnatal day 21 (P21) and tests for neurobehavioral toxicity were performed from P3 to P21. Hypoxic-ischemic insults resulted in severe white matter injury, enlarged ventricles, deficits in the hippocampus, reduction in numbers of mature oligodendrocytes and tyrosine hydroxylase-positive neurons, damage to axons and dendrites, and impaired myelination, as indicated by the decrease in myelin basic protein immunostaining in the P21 rat brain. Hypoxic-ischemic insult also significantly affected physical development (body weight gain and eye opening) and neurobehavioral performance, including sensorimotor and locomotor function, anxiety and cognitive ability in the P21 rat. Treatments with minocycline significantly attenuated the hypoxia-ischemia-induced brain injury and improved neurobehavioral performance. The protection of minocycline was associated with its ability to reduce microglial activation. The present results show that minocycline has long-lasting protective effects in the neonatal rat brain in terms of both hypoxia-ischemia-induced brain injury and the associated neurological dysfunction.

Topics: Age Factors; Animals; Animals, Newborn; Brain; Brain Damage, Chronic; Brain Infarction; Cytoprotection; Disease Models, Animal; Female; Fetal Hypoxia; Gliosis; Humans; Hypoxia-Ischemia, Brain; Infant, Newborn; Injections, Intraperitoneal; Leukomalacia, Periventricular; Male; Minocycline; Nerve Degeneration; Nerve Fibers, Myelinated; Neuroprotective Agents; Rats; Rats, Sprague-Dawley; Treatment Outcome

Hypoxic-ischemic encephalopathy (HIE) is a leading cause of mortality and morbidity during the perinatal period, and currently no therapeutic drug is available. Minocycline, an antibiotic, has recently been shown to have neuroprotective effects distinct from its antimicrobial effect in several neurological disorders including ischemic brain injury. We examined the effect of minocycline on neonatal hypoxic-ischemic brain injury by using histologic scoring in both mouse and rat models. Mouse (C57Bl/6) and rat (SD) pups were exposed to a unilateral hypoxic-ischemic insult at 8 and 7 days of age, respectively. Minocycline hydrochloride was administered according to protocols that were reported to provide neuroprotection in adult or neonatal rats. Seven days after the insult, we examined brain injury in Nissl stained sections. Although minocycline ameliorated brain injury in the developing rat, it increased injury in the developing mouse. This detrimental effect in the mouse was consistent across different regions (cortex, striatum, and thalamus), with both single and multiple injection protocols and with both moderate and high-dose treatment (P < 0.05). The mechanism of the contrasting effects in mouse and rat is not clear and remains to be elucidated. Minocycline has been used as an antibiotic in the clinical setting for decades; therefore, it may be considered for use in infants with hypoxic-ischemic brain damage, based on prior reports of neuroprotection in the rat. However, it is important to examine this drug carefully before clinical use in human infants, taking our data in the mouse model into consideration.

Topics: Analysis of Variance; Animals; Animals, Newborn; Brain Infarction; Brain Ischemia; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Administration Schedule; Female; Hypoxia-Ischemia, Brain; Male; Mice; Mice, Inbred C57BL; Minocycline; Random Allocation; Rats; Sensitivity and Specificity; Statistics, Nonparametric

Hypoxic-ischemic brain injury in the perinatal period is a major cause of morbidity and mortality. Presently, there are no proven effective therapies with which to safeguard the human neonatal brain against this type of injury. Minocycline, a semisynthetic tetracycline, has been shown to be neuroprotective in certain adult ischemic injury/stroke and neurodegenerative disease models. However, minocycline's neuroprotective effects have not been assessed after insults to the neonatal brain. We now report that minocycline administered either immediately before or immediately after a hypoxic-ischemic insult substantially blocks tissue damage in a rodent model of neonatal hypoxic-ischemic brain injury. Minocycline treatment prevents the formation of activated caspase-3, a known effector of apoptosis, as well as the appearance of a calpain cleaved substrate, a marker of excitotoxic/necrotic cell death. To our knowledge, this is the first report of a systemic treatment that can be administered after a hypoxic-ischemic insult, which provides robust, nearly complete neuroprotection to the developing brain. Our data suggest that minocycline or a related neuroprotective tetracycline may be a candidate to consider in human clinical trials to protect the developing brain against hypoxic-ischemic-induced damage.

Topics: Animals; Animals, Newborn; Anti-Bacterial Agents; Brain; Carotid Arteries; Caspase 3; Caspase Inhibitors; Caspases; Hypoxia-Ischemia, Brain; Injections, Intraperitoneal; Ligation; Minocycline; Rats; Rats, Sprague-Dawley