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Disability or death due to intracerebral hemorrhage (ICH) is attributed to blood lysis, liberation of iron, and consequent oxidative stress. Iron chelators bind to free iron and prevent neuronal death induced by oxidative stress and disability due to ICH, but the mechanisms for this effect remain unclear. We show that the hypoxia-inducible factor prolyl hydroxylase domain (HIF-PHD) family of iron-dependent, oxygen-sensing enzymes are effectors of iron chelation. Molecular reduction of the three HIF-PHD enzyme isoforms in the mouse striatum improved functional recovery after ICH. A low-molecular-weight hydroxyquinoline inhibitor of the HIF-PHD enzymes, adaptaquin, reduced neuronal death and behavioral deficits after ICH in several rodent models without affecting total iron or zinc distribution in the brain. Unexpectedly, protection from oxidative death in vitro or from ICH in vivo by adaptaquin was associated with suppression of activity of the prodeath factor ATF4 rather than activation of an HIF-dependent prosurvival pathway. Together, these findings demonstrate that brain-specific inactivation of the HIF-PHD metalloenzymes with the blood-brain barrier-permeable inhibitor adaptaquin can improve functional outcomes after ICH in several rodent models., (Copyright © 2016, American Association for the Advancement of Science.)

Background: Brain injury after intracerebral hemorrhage (ICH) arises from numerous contributors, of which some also play essential roles. Notably, thrombin production, needed to stop bleeding, also causes acute cell death and edema. In some rodent models of ICH, peri-hematoma neurons die over weeks. Hence we evaluated whether thrombin is responsible for this chronic degeneration. Functional impairments after ICH also result from sub-lethal damage to neurons, especially the loss of dendrites. Thus, we evaluated whether thrombin infusion alone, a reductionist model of ICH, causes similar injury., Methods: Adult rats had a modest intra-striatal infusion of thrombin (1 U) or saline followed by a behavioral test, to verify impairment, 7 days later. After this they were euthanized and tissue stained with Golgi-Cox solution to allow the assessment of dendritic morphology in striatal neurons. In a second experiment, rats survived 7 or 60 days after thrombin infusion in order to histologically determine lesion volume., Results: Thrombin caused early cell death and considerable atrophy in surviving peri-lesion neurons, which had less than half of their usual numbers of branches. However, total tissue loss was comparable at 7 (24.1 mm3) and 60 days (25.6 mm3)., Conclusion: Thrombin infusion causes early cell death and neuronal atrophy in nearby surviving striatal neurons but thrombin does not cause chronic tissue loss. Thus, the chronic degeneration found after ICH in rats is not simply and solely due to acute thrombin production. Nonetheless, thrombin is an important contributor to behavioral dysfunction because it causes cell death and substantial dendritic injury.

Intracerebral hemorrhage (ICH) is a devastating stroke causing considerable tissue destruction from mechanical trauma and secondary degeneration. Free iron, released over days from degrading erythrocytes, causes free radicals that likely contribute to delayed injury. Indeed, an intracerebral injection of iron rapidly kills cells and causes cerebral edema. We expanded upon these observations by: determining a dose-response relationship of iron infusion, examining the structural appearance of surviving striatal neurons, and evaluating injury over months. First, we measured 24-h edema in rats given 3.8, 19.0 or 38.0 μg infusions of FeCl₂ (i.e., 30 μL of a 1, 5 or 10 mmol/L solution). Second, rats were given these infusions (vs. saline controls) followed by behavioral assessment and histology at 7 days. Third, dendritic structure was measured in Golgi-Cox stained neurons at 7 days after a 0.95-μg dose (30 μL of a 0.25 mmol/L solution). Last, rats survived 7 or 60 days post-injection (19.0 μg) for histological assessment. Larger doses of iron caused greater injury, but this was generally not reflected in behavior that indicated similar deficits among the 3.8-38.0 μg groups. Similarly, edema occurred but was not linearly related to dose. Even after a low iron dose the surviving neurons in the peri-injury zone were considerably atrophied (vs. contralateral side and controls). Finally, continuing tissue loss occurred over weeks with prominent neuronal death and iron-positive cells (e.g., macrophages) at 60 days. Iron alone may account for the chronic degeneration found after ICH in rodent models., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Delayed hypothermia salvages CA1 neurons from global ischemic injury. However, the effects of this potent neuroprotectant on endogenous repair mechanisms, such as neurogenesis, have not been clearly examined. In this study, we quantified and phenotyped newly generated cells within the hippocampus following untreated and hypothermia-treated ischemia. We first show that CA1 pyramidal neurons did not spontaneously regenerate after ischemia. We then compared the level of neuroprotection when hypothermia was initiated either during or after ischemia. Treatment efficacy decreased with longer delays, but hypothermia delayed for up to 12 hours was neuroprotective. Although bromodeoxyuridine (BrdU) incorporation was elevated in ischemic groups, CA1 neurogenesis did not occur as the BrdU label did not colocalize with neuronal nuclei (NeuN) in any of the groups. Instead, the majority of BrdU-labeled cells were Iba-positive microglia, and neuroprotective hypothermia decreased the delayed generation of microglia during the third postischemic week. Conversely, hypothermia delayed for 12 hours significantly increased the survival of newly generated dentate granule cells at 4 weeks after ischemia. Thus, our findings show that CA1 neurogenesis does not contribute to hypothermic neuroprotection. Importantly, we also show that prolonged hypothermia positively interacts with postischemic repair processes, such as neurogenesis, resulting in improved functional outcome.

Background and Purpose: Recent studies show that prolonged (eg, 24-hour) postischemic hypothermia confers lasting histological and behavioral protection against severe global cerebral ischemia. However, functional abnormalities may be compensated for by undamaged brain regions and thus not detected by behavioral tests. To determine whether hypothermia preserves CA1 functional integrity, we measured synaptic and membrane properties of CA1 neurons in ischemic gerbils treated with postischemic hypothermia., Methods: Gerbils were subjected to 5 minutes of forebrain ischemia and were either left untreated or exposed to 2 days of hypothermia (32 degrees C for 24 hours and then 34 degrees C for 24 hours). Sham animals were operated on but not made ischemic, then either allowed to recover at room temperature or subjected to hypothermia for 2 days. Approximately 5 weeks after ischemia or sham surgery, patch-clamp recordings were obtained from the CA1 region of hippocampal slices., Results: There was approximately 95% CA1 cell loss in untreated ischemic animals, whereas ischemic gerbils treated with hypothermia had cell counts similar to sham animals. Resting membrane potential, action potential amplitude and duration, input resistance, and synaptic currents evoked by Schaffer collateral stimulation were similar between pyramidal cells obtained from ischemic gerbils treated with hypothermia and sham-operated animals (P:>0.05)., Conclusions: These data demonstrate that postischemic hypothermia preserves the measured electrophysiological properties of CA1 neurons in the absence of any apparent functional abnormalities. This study provides further support for the use of hypothermia as a treatment for cerebral ischemia.

It has been repeatedly claimed that neuronal death in the hippocampal CA1 sector after untreated global ischemia occurs via apoptosis. This is based largely on DNA laddering, nick end labeling, and light microscopy. Delineation of apoptosis requires fine structural examination to detect morphological events of cell death. We studied the light and ultrastructural characteristics of CA1 injury after 5 min of untreated global ischemia in gerbils. To increase the likelihood of apoptosis, some ischemic gerbils were subjected to delayed postischemic hypothermia, a treatment that mitigates injury and delays the death of some neurons. In these gerbils, 2 d of mild hypothermia was initiated 1, 6, or 12 hr after ischemia, and gerbils were killed 4, 14, or 60 d later. Ischemia without subsequent cooling killed 96% of CA1 neurons by day 4, whereas all hypothermia-treated groups had significantly reduced injury at all survival times (2-67% loss). Electron microscopy of ischemic neurons with or without postischemic hypothermia revealed features of necrotic, not apoptotic, neuronal death even in cells that died 2 months after ischemia. Dilated organelles and intranuclear vacuoles preceded necrosis. Unique to the hypothermia-treated ischemic groups, some salvaged neurons were persistently abnormal and showed accumulation of unusual, morphologically complex secondary lysosomes. These indicate selective mitochondrial injury, because they were closely associated with normal and degenerate mitochondria, and transitional forms between mitochondria and lysosomes occurred. The results show that untreated global ischemic injury has necrotic, not apoptotic, morphology but do not rule out programmed biochemical events of the apoptotic pathway occurring before neuronal necrosis.

The effects of temperature and scopolamine on dizocilpine maleate-induced neuronal necrosis in the rat cingulate/retrosplenial cortex, entorhinal/olfactory cortices and the dentate gyrus were studied. Mild, protracted hypothermia (48 h at a brain temperature of 34 degrees C), induced by a servo-controlled "exposure technique" in the awake female rat, significantly reduced dizocilpine maleate (5.0 mg/kg, i.p.)-induced neuronal death in the cingulate/retrosplenial and entorhinal/olfactory cortices seven days following drug administration. Scopolamine (0.25 mg/kg, i.p.), putatively neuroprotective [Olney J. W. et al. (1991) Science 254, 1515-1518], did not reduce injury in the cingulate/retrosplenial cortex of female rats following one injection, but did following two and three doses. Scopolamine had no significant effect in the other brain regions. A temperature elevation of only 1 degree C above baseline for 48 h in awake female rats increased dizocilpine maleate-induced damage. Finally, the sex differences in N-methyl-D-aspartate antagonist toxicity were replicated and extended to other structures, and found not to be due to temperature differences. Our data show that dizocilpine maleate neurotoxicity is temperature sensitive. Scopolamine treatment needed to be prolonged in order to reduce injury, and even then was only efficacious in one of three brain regions. The results underscore the importance of using neuronal necrosis in several brain regions as the endpoint and for the use of prolonged therapeutic interventions. Furthermore, given the potential hypothermic action of other putative neuroprotective drugs, a mechanistic re-evaluation of N-methyl-D-aspartate antagonist-induced injury is needed, with precise brain temperature measurement.

Five minutes of global ischemia in gerbil results in delayed hippocampal CA1 neuronal degeneration, which is accompanied by working memory impairments and hyperactivity in novel environments. In this study, postischemic activity was characterized in familiar and in novel environments to determine whether hyperactivity was due to impaired spatial habituation or another form of motor hyperactivity. This study also determined whether 6-h delayed hypothermia, which reduces CA1 neuronal injury, would attenuate functional impairments. Gerbils were subjected to 5 min of normothermic ischemia or sham operation 2 days following implantation of brain temperature probes. One of two ischemic groups was cooled (>48 h) starting at 6-h postischemia. Locomotor activity in a familiar cage was measured for 6 days while activity in three novel environments was intermittently measured on days 4, 5 and 6. Open field behavior and working memory in a T-maze were also assessed. Untreated ischemia caused marked hyperactivity in the familiar cage on day 1, which reverted to near-normal by day 2. Nonetheless, these gerbils showed hyperactivity during novel environment sessions on days 4-6. This maze behavior, which predicted hippocampal CA1 injury, was not due to different habituation rates nor baseline hyperactivity. Conversely, open field sessions on day 8 revealed ischemic habituation rate deficits. Ischemia also impaired working memory in the T-maze. Delayed hypothermia, which reduced neuronal loss in the CA1 sector to 12% from 81%, reduced all functional impairments. Ischemic gerbils quickly developed spontaneous locomotion hyperactivity that returned to near-normal after 1 day. This motor hyperactivity did not explain the elevated activity found with delayed testing in novel environments. Regardless, only the open field test on day 8 revealed a habituation-like deficit., (Copyright 1998 Elsevier Science B.V.)

Global ischemia, in the gerbil, produces profound hippocampal CA1 loss which leads to functional abnormalities (e.g. habituation impairment). In experiment 1, gerbils were subjected to 3 or 5 min of normothermic (brain) ischemia. Hypothermic groups were cooled to 32 degrees C for 12 h beginning 1 h after ischemia, while control groups (no hypothermia) regulated their own temperature. Exploration in a novel open field was assessed on days 3, 7 and 10 following ischemia and CA1 neurons were counted after 10- or 30-day survival. Both ischemia durations produced severe CA1 necrosis which resulted in increased open field activity. Hypothermia attenuated this behavioral pattern and substantially reduced CA1 necrosis against 3 min of ischemia when assessed at 10 and 30 days, but was only partially effective against a 5 min occlusion where, in addition, some cell death appeared to be delayed rather than prevented. In experiment 2, gerbils were occluded for 5 min and survived for 30 days. Twenty-four hours of hypothermia initiated 1 h after ischemia resulted in near total preservation of CA1 neurons. Thus, increasing the duration of post-ischemic hypothermia from 12 to 24 h produced much greater neuroprotection against severe ischemia. Prolonged post-ischemic hypothermia may be a valuable intervention in stroke patients.