Effects of low-dose unfractionated heparin on early brain injury after subarachnoid hemorrhage in mice.

BACKGROUND: Sphingosine kinase (SphK) 1 has been reported as an important signaling node in anti-apoptotic signaling. Heparin is a pleiotropic drug that antagonizes many pathophysiological mechanisms. In this study, we evaluated if heparin prevents early brain injury (EBI) after subarachnoid hemorrhage (SAH) by anti-apoptotic mechanisms including SphK1. METHODS: SAH was induced by endovascular perforation in mice, which were randomly assigned to sham-operated (n = 23), SAH + vehicle (n = 36), SAH + 10U heparin pretreatment (n = 13), SAH + 30U heparin pretreatment (n = 15), SAH + 10U heparin posttreatment (n = 31), and SAH + 30U heparin posttreatment (n = 23). At 24 hours post-SAH, neurological scores, brain water content and Evans blue extravasation were evaluated. Also, the expression of SphK, phosphorylated Akt, and cleaved caspase-3 was determined by Western blotting, and cell death was examined by terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end-labeling staining. RESULTS: Low-dose heparin posttreatment improved neurobehavioral function, brain edema, blood-brain barrier disruption and cell death in the cortex, associated with an increase in SphK1 and phosphorylated Akt, and a decrease in cleaved caspase-3. High-dose heparin had a tendency for increased SAH severity, which obscured the neuroprotective effects by heparin. CONCLUSIONS: Low-dose heparin posttreatment may decrease the development of post-SAH EBI through anti-apoptotic mechanisms including sphingosine-related pathway activation.

The arousal effect of hyperbaric oxygen through orexin/hypocretin an upregulation on ketamine/ethanol-induced unconsciousness in male rats.

Approaches that facilitate the recovery from coma would have enormous impacts on patient outcomes and medical economics. Orexin-producing neurons release orexins (also known as hypocretins) energy-dependently to maintain arousal. Hyperbaric oxygen (HBO) could increase ATP levels by preserving mitochondrial function. We investigated, for the first time, the arousal effects of HBO and orexins mechanisms in a rat model of unconsciousness induced by ketamine or ethanol. A total of 120 Sprague-Dawley male rats were used in this study. Unconsciousness was induced either by intraperitoneal injection of ketamine or ethanol. The HBO treatment (100% O2 at 3 ATA) was administered immediately after unconsciousness induction for 1 hr. SB334867, orexin-1 receptor (OX1R) inhibitor, or JNJ10397049, orexin-2 receptor (OX2R) inhibitor was administered 30 min intraperitoneally before unconsciousness induction. Loss of righting reflex test (LORR) and Garcia test were used to evaluate the unconsciousness duration and neurological deficits after recovering from unconsciousness, respectively. Enzyme-linked immunosorbent assay was used to measure brain tissue ATP and orexin A levels. Ketamine or ethanol injection resulted in LORR immediately and neurological deficits 6 hr after unconsciousness induction. HBO treatment significantly reduced the LORR duration, improved Garcia scores and unregulated ATP and orexin A levels in the brain tissue. Administration of OX1R inhibitor or OX2 R inhibitor abolished arousal and neurological benefits of HBO. In conclusion, HBO exerted arousal-promoting effects on unconscious rats induced by ketamine or ethanol. The underlying mechanism was via, at least in part, ATP/orexin A upregulation. HBO may be a practical clinical approach to accelerate unconsciousness recovery in patients.

Hydrogen gas therapy improves survival rate and neurological deficits in subarachnoid hemorrhage rats: a pilot study.

The high morbidity, high mortality, and significant shortage of effective therapies for subarachnoid hemorrhage (SAH) have created an urgency to discover novel therapies. Human studies in Asia have established the safety of hydrogen gas in the treatment of hepatic, renal, pulmonary, and cardiac diseases. Mechanistically, hydrogen gas has been shown to affect oxidative stress, inflammation, and apoptosis. We hypothesized that hydrogen therapy would improve neurological function and increase survival rate in SAH. High dose hydrogen gas (66% at 3 L/min) was administered for 2 hours at 0.5, 8, and 18 hours after SAH. This treatment increased 72-hour survival rate and provided 24-hour neuroprotection after SAH in rats. To our knowledge, this is the first report demonstrating that high dose hydrogen gas therapy reduces mortality and improves outcome after SAH. Our results correlate well with the proposed mechanisms of hydrogen gas therapy within the literature. We outline four pathways and downstream targets of hydrogen gas potentially responsible for our results. A potentially complex network of pathways responsible for the efficacy of hydrogen gas therapy, along with a limited mechanistic understanding of these pathways, justifies further investigation to provide a basis for clinical trials and the advancement of hydrogen gas therapy in humans. This study was approved by the Institutional Animal Care and Use Committee of Loma Linda University, USA (Approval No. 8160016) in May 2016.

Developing a standardized system of exposure and intervention endpoints for isoflurane in preclinical stroke models.

Isoflurane is a regularly used anesthetic in translational research. Isoflurane facilitates invasive surgery and a rapid recovery. Specifically, in the pathology of stroke, controversy has surrounded isoflurane's intrinsic neuroprotective abilities, affecting apoptosis, excitotoxicity, and blood brain barrier disruption. Due to the intrinsic neuroprotective nature and lack of standardized guidelines for the use of isoflurane, research has shifted away from this gas in most animal models. Antagonistically, studies have also reported that no neuroprotective effects are observed when a surgery is accompanied with isoflurane exposure under 20 minutes. Isoflurane affects the pathophysiology in stroke patients by altering critical pathways in endothelial, neuronal, and microglial cells. Current studies have elucidated isoflurane neuroprotection to be time dependent and may be minimized in experimental designs if the exposure time is limited to a specific window. Therefore, with detailed and extensive literature on anesthetics, we can hypothesize that isoflurane exposure under the 20-minute benchmark, behavior and molecular pathways can be evaluated at any time-point following ischemic insult without confounding artifacts from isoflurane; however, If the exposure to isoflurane exceeds 20 minutes, the acute neuroprotective effects are evident for 2 weeks in the model, which should be accounted for in molecular and behavioral assessments, with either isoflurane inhibitors or a control group at 2 weeks post middle cerebral artery occlusion. The purpose of this review is to suggest a detailed and standardized outline for interventions and behavioral assessments after the use of isoflurane in experimental designs.

Hyperbaric Oxygen Reduces Infarction Volume and Hemorrhagic Transformation Through ATP/NAD+/Sirt1 Pathway in Hyperglycemic Middle Cerebral Artery Occlusion Rats.

BACKGROUND AND PURPOSE: Energy depletion is a critical factor leading to cell death and brain dysfunction after ischemic stroke. In this study, we investigated whether energy depletion is involved in hyperglycemia-induced hemorrhagic transformation after ischemic stroke and determined the pathway underlying the beneficial effects of hyperbaric oxygen (HBO). METHODS: After 2-hour middle cerebral artery occlusion, hyperglycemia was induced by injecting 50% dextrose (6 mL/kg) intraperitoneally at the onset of reperfusion. Immediately after it, rats were exposed to HBO at 2 atmospheres absolutes for 1 hour. ATP synthase inhibitor oligomycin A, nicotinamide phosphoribosyl transferase inhibitor FK866, or silent mating type information regulation 2 homolog 1 siRNA was administrated for interventions. Infarct volume, hemorrhagic volume, and neurobehavioral deficits were recorded; the level of blood glucose, ATP, and nicotinamide adenine dinucleotide and the activity of nicotinamide phosphoribosyl transferase were monitored; the expression of silent mating type information regulation 2 homolog 1, acetylated p53, acetylated nuclear factor-κB, and cleaved caspase 3 were detected by Western blots; and the activity of matrix metalloproteinase-9 was assayed by zymography. RESULTS: Hyperglycemia deteriorated energy metabolism and reduced the level of ATP and nicotinamide adenine dinucleotide and exaggerated hemorrhagic transformation, blood-brain barrier disruption, and neurological deficits after middle cerebral artery occlusion. HBO treatment increased the levels of the ATP and nicotinamide adenine dinucleotide and consequently increased silent mating type information regulation 2 homolog 1, resulting in attenuation of hemorrhagic transformation, brain infarction, as well as improvement of neurological function in hyperglycemic middle cerebral artery occlusion rats. CONCLUSIONS: HBO induced activation of ATP/nicotinamide adenine dinucleotide/silent mating type information regulation 2 homolog 1 pathway and protected blood-brain barrier in hyperglycemic middle cerebral artery occlusion rats. HBO might be promising approach for treatment of acute ischemic stroke patients, especially patients with diabetes mellitus or treated with r-tPA (recombinant tissue-type plasminogen activator).

Hyperbaric oxygen therapy for traumatic brain injury: bench-to-bedside.

Traumatic brain injury (TBI) is a serious public health problem in the United States. Survivors of TBI are often left with significant cognitive, behavioral, and communicative disabilities. So far there is no effective treatment/intervention in the daily clinical practice for TBI patients. The protective effects of hyperbaric oxygen therapy (HBOT) have been proved in stroke; however, its efficiency in TBI remains controversial. In this review, we will summarize the results of HBOT in experimental and clinical TBI, elaborate the mechanisms, and bring out our current understanding and opinions for future studies.

Hyperbaric Oxygen Preconditioning Attenuates Hemorrhagic Transformation Through Reactive Oxygen Species/Thioredoxin-Interacting Protein/Nod-Like Receptor Protein 3 Pathway in Hyperglycemic Middle Cerebral Artery Occlusion Rats.

OBJECTIVES: To clarify whether hyperbaric oxygen preconditioning can attenuate hyperglycemia-enhanced hemorrhagic transformation and to establish a role for Nod-like receptor protein 3 inflammasome in the pathophysiology of hemorrhagic transformation. DESIGN: Controlled prospective animal study. SETTING: University research laboratory. SUBJECTS: Male Sprague-Dawley rats weighing 260-280 g. INTERVENTIONS: Rats received 1-hour-long hyperbaric oxygen preconditioning for five consecutive days. Hyperglycemic middle cerebral artery occlusion model was induced at 24 hours after the last hyperbaric oxygen exposure. Reactive oxygen species scavenger (N-acetyl-L-cysteine), thioredoxin-interacting protein small interfering RNA, and Nod-like receptor protein 3 small interfering RNA were given in different groups separately to verify the possible pathway. MEASUREMENTS AND MAIN RESULTS: Rats were randomly divided into sham, middle cerebral artery occlusion, middle cerebral artery occlusion + dextrose, middle cerebral artery occlusion + dextrose + normobaric oxygen preconditioning, middle cerebral artery occlusion + dextrose + hyperbaric oxygen, middle cerebral artery occlusion + dextrose + hyperbaric oxygen + N-acetyl-L-cysteine, middle cerebral artery occlusion + dextrose + hyperbaric oxygen + control small interfering RNA, middle cerebral artery occlusion + dextrose + hyperbaric oxygen + thioredoxin-interacting protein small interfering RNA, and middle cerebral artery occlusion + dextrose + hyperbaric oxygen + Nod-like receptor protein 3 small interfering RNA groups. Hyperglycemia was induced by administration of 50% dextrose (6 mL/kg) intraperitoneally 30 minutes before middle cerebral artery occlusion. Control small interfering RNA/thioredoxin-interacting protein small interfering RNA or Nod-like receptor protein 3 small interfering RNA (500 pmol/5 µL) were injected intracerebroventricularly 72 hours before middle cerebral artery occlusion for intervention. The neurologic scores, infarction and hemorrhage volumes, the expression of Nod-like receptor protein 3, and its downstream targets were analyzed. Hyperbaric oxygen preconditioning decreased both infarction and hemorrhage volumes and improved neurobehavioral function. In addition, hyperbaric oxygen preconditioning provided additional protective effects in hemorrhagic transformation, which was independent of infarction volume. The benefits of hyperbaric oxygen preconditioning on hyperglycemic middle cerebral artery occlusion rats were reversed after blocking the reactive oxygen species/thioredoxin-interacting protein/Nod-like receptor protein 3 pathway. CONCLUSIONS: Nod-like receptor protein 3 inflammasome played an important role in hyperglycemia-enhanced hemorrhagic transformation. Hyperbaric oxygen preconditioning attenuated hemorrhagic transformation through reactive oxygen species/thioredoxin-interacting protein/Nod-like receptor protein 3 pathway.

Hyperbaric oxygen therapy for post concussion symptoms: issues may affect the results.

Post concussion syndrome (PCS) is a set of symptoms succeeding in 25 % of mild traumatic brain injury (mTBI) patients. Hyperbaric oxygen therapy (HBOT) has been demonstrated as an effective method for treating acute and severe TBI, but its efficacy in PCS remains controversial. In this editorial, we reviewed the clinical studies of HBOT in PCS, summarized the limitations of these studies, and discussed the limitations: inappropriate Sham group using room air at 1.2 or 1.3 ATA; delayed HBO administration; subjective assessment methods; time point for outcome assessment and small sample size. We hope that our concerns will be helpful for future clinical studies of HBO therapy in TBI or other neurological disorders.

Hyperbaric oxygen preconditioning attenuates hemorrhagic transformation through increasing PPARγ in hyperglycemic MCAO rats.

Hyperbaric oxygen preconditioning (HBO-PC) has been demonstrated to attenuate hemorrhagic transformation (HT) after middle cerebral artery occlusion (MCAO) in hyperglycemic rats. However, the mechanisms remain to be illustrated. Recently, HBO-PC has been shown to activate peroxisome proliferator-activated receptor-gamma (PPARγ) by increasing 15d-PGJ2 in primary cultured neurons. We hypothesize that HBO-PC reduces HT by suppressing inflammation through increasing 15d-PGJ2 and activating PPARγ in hyperglycemic MCAO rats. HBO (2.5ATA) was administered for 1h daily for 5 consecutive days. The PPARγ inhibitor GW9662 was administered intraperitoneally to designated animals. Infarction volume, hemorrhage volume, neurological scores and mortality were analyzed. The levels of 15d-PGJ2, PPARγ, TNF-α and IL-1ß, tight junction proteins as well as the activity of MMP-2 and MMP-9 were evaluated 24h after MCAO. HBO-PC reduced HT, improved neurological function, down-regulated inflammatory molecules and inhibited the activation of MMP-9 by increasing 15d-PGJ2 and PPARγ at 24h after MCAO. The results suggested that HBO-PC might be an alternative measure to decrease HT in ischemic stroke.

Hyperbaric oxygen therapy fails to reduce hydrocephalus formation following subarachnoid hemorrhage in rats.

BACKGROUND & PURPOSE: Approximately 40% of hemorrhagic stroke survivors develop hydrocephalus. Hyperbaric oxygen (HBO) has been shown to be anti-inflammation following experimental stroke; however, its effect upon post-hemorrhagic hydrocephalus formation is not known. The objective of this study is to investigate whether HBO therapy can effectively reduce hydrocephalus formation and improve neurobehavioral functions in a rat model of subarachnoid hemorrhage (SAH). METHOD: Thirty-eight male Sprague-Dawley rats (300-320 g) rats survived for 21 days from SAH by endovascular perforation or sham surgery were used. At 24 hours after SAH, HBO (3 atmospheres absolute) or normobaric oxygen (NBO) administrated for 1 hour once daily for a total of 7 days. Wire hanging and rotarod testing were conducted at 14 days after SAH, and cognitive functions were evaluated via the Morris water maze, between day 17 to day 21 after surgery. At day 21, rats were sacrificed and cerebroventricular volumes were measured histologically. RESULTS: Hydrocephalus exacerbated neurological deficits after SAH, and HBO multiple treatment tendentially improved the neurobehavioral functions. Spatial learning and memory deficits were noticed after SAH, and rats with hydrocephalus showed worse learning and memory abilities and HBO treatment showed a minor improvement. In the SAH group (room air) 4 rats showed an increased ventricular volume at day 21 after SAH-induction (n = 10). HBO or NBO therapy did not alter the occurrence of hydrocephalus after SAH, as 4 rats in each of these groups showed an increased ventricular volume (n = 10 per group). CONCLUSION: Multiple HBO therapy does not ameliorate hydrocephalus formation in a rat model of SAH; however, HBO tendentially improved the neurological functions and spatial learning and memory abilities in rats with hydrocephalus.

Delayed hyperbaric oxygen therapy promotes neurogenesis through reactive oxygen species/hypoxia-inducible factor-1α/ß-catenin pathway in middle cerebral artery occlusion rats.

BACKGROUND AND PURPOSE: Hyperbaric oxygen (HBO) has been reported to be neuroprotective and to improve neurofunctional outcomes in acute stroke. However, it is not clear whether delayed HBO enhances endogenous neurogenesis and promotes neurofunctional recovery. The aim of this study is to evaluate the effects of delayed HBO therapy on neurogenesis and its potential mechanisms. METHODS: One hundred eleven male Sprague-Dawley rats that survived for 7 days from 2 hours of middle cerebral artery occlusion and reperfusion were used. Delayed and multiple HBO were administrated beginning at 7 days after middle cerebral artery occlusion and lasting for 42 days with 3 HBO-free intervals (5 days each). Motor sensory deficits were measured by foot-fault test, and learning and memory abilities were evaluated by Morris water maze. Neurogenesis was examined by double immunostaining of bromodeoxyuridine and doublecortin, bromodeoxyuridine and neuronal nuclei at day 42. For mechanism studies, inhibitors for reactive oxygen species (ROS), hypoxia-inducible factor (HIF)-1α, and ß-catenin were administrated, and the levels of ROS, HIF-1α, ß-catenin, lymphoid enhancer-binding factor-1, T-cell factor-1, neurogenin-1, doublecortin, and synapsin-1 were assessed by ELISA or Western blot at day 14. RESULTS: Delayed HBO treatment promoted neurogenesis and improved neurofunctional recovery at day 42, and the improvements were reversed by inhibition of ROS and HIF-1α. Delayed HBO significantly increased ROS and HIF-1α, and upregulated the expression of neurogenin-1, doublecortin, and synapsin-1. Inhibition of ROS and HIF-1α removed the effects of delayed HBO. CONCLUSIONS: Delayed HBO enhanced endogenous neurogenesis and improved neurofunctional recovery in the late-chronic phase of stroke possibly mediated by ROS/HIF-1α/ß-catenin pathway. Delayed HBO may serve as an alternative treatment to improve long-term recovery of stroke survivors.

Correlation between subacute sensorimotor deficits and brain edema in two mouse models of intracerebral hemorrhage.

Formation of brain edema after intracerebral hemorrhage (ICH) is highly associated with its poor outcome. However, the relationship between cerebral edema and behavioral deficits has not been thoroughly examined in the preclinical setting. Hence, this study aimed to evaluate the ability of common sensorimotor tests to predict the extent of brain edema in two mouse models of ICH. One hundred male CD-1 mice were subjected to sham surgery or ICH induction via intrastriatal injection of either autologous blood (30 µL) or bacterial collagenase (0.0375U or 0.075U). At 24 and 72 h after surgery, animals underwent a battery of behavioral tests, including the modified Garcia neuroscore (Neuroscore), corner turn test (CTT), forelimb placing test (FPT), wire hang task (WHT) and beam walking (BW). Brain edema was evaluated via the wet weight/dry weight method. Intrastriatal injection of autologous blood or bacterial collagenase resulted in a significant increase in brain water content and associated sensorimotor deficits (p<0.05). A significant correlation between brain edema and sensorimotor deficits was observed for all behavioral tests except for WHT and BW. Based on these findings, we recommend implementing the Neuroscore, CTT and/or FPT in preclinical studies of unilateral ICH in mice.

Hyperbaric oxygen preconditioning attenuates hyperglycemia-enhanced hemorrhagic transformation by inhibiting matrix metalloproteinases in focal cerebral ischemia in rats.

Hyperglycemia dramatically aggravates brain infarct and hemorrhagic transformation (HT) after ischemic stroke. Oxidative stress and matrix metalloproteinases (MMPs) play an important role in the pathophysiology of HT. Hyperbaric oxygen preconditioning (HBO-PC) has been proved to decrease oxidative stress and has been demonstrated to be neuroprotective in experimental stroke models. The present study determined whether HBO-PC would ameliorate HT by a pre-ischemic increase of reactive oxygen species (ROS) generation, and a suppression of MMP-2 and MMP-9 in hyperglycemic middle cerebral artery occlusion (MCAO) rats. Rats were pretreated with HBO (100% O2, 2.5 atmosphere absolutes) 1 h daily for 5 days before MCAO. Acute hyperglycemia was induced by an injection of 50% dextrose. Neurological deficits, infarction volume and hemorrhagic volume were assessed 24 h and 7 days after ischemia. ROS scavenger n-acetyl cysteine (NAC), hypoxia-inducible factor-1α (HIF-1α), inhibitor 2-methoxyestradiol (2ME2) and activator cobalt chloride (CoCl2), and MMP inhibitor SB-3CT were administrated for mechanism study. The activity of MMP-2 and MMP-9, and the expression HIF-1α were measured. HBO-PC improved neurological deficits, and reduced hemorrhagic volume; the expression of HIF-1α was significantly decreased, and the activity of MMP-2 and MMP-9 was reduced by HBO-PC compared with vehicle group. Our results suggested that HBO-PC attenuated HT via decreasing HIF-1α and its downstream MMP-2 and MMP-9 in hyperglycemic MCAO rats.

Serum leptin levels decrease after permanent MCAo in the rat and remain unaffected by delayed hyperbaric oxygen therapy.

Hyperbaric oxygen therapy (HBOT), referring to the medical use of oxygen at a level higher than atmospheric pressure, exerts neuroprotective effects after ischemic stroke via various mechanisms. It has been demonstrated that HBOT modulates the synthesis and degradation of hormones. Leptin, an adipose derived hormone, has been found to confer neuroprotection following experimental stroke. However, it is not known whether HBOT alters leptin concentrations after permanent middle cerebral artery occlusion (pMCAo) in the rat. In this present study, we aimed to investigate the effect of HBOT on the serum concentration of leptin in rats subjected to pMCAo. HBOT was initiated 48 hrs after experimental pMCAo, at 2.5 atmospheres absolutes with 100% oxygen, 1 hr a day for 10 consecutive days. Body weight, neurobehavioral deficits and infarct size were evaluated. Blood was collected on day 1 and day 16 following HBOT. Serum leptin concentrations were measured with ELISA. Delayed HBOT reduced infarct size and improved neurobehavioral scores. Decreased serum levels of leptin were found in treated and untreated pMCAo animals, compared to the sham group on day 1 (P > 0.05) and day 16 (P < 0.05). However, no statistical significance was found between HBOT and the air group. We concluded that the neuroprotective effects of delayed HBOT in pMCAo rats were unlikely to be exerted through changes in the serum concentration of leptin.

Fingolimod reduces cerebral lymphocyte infiltration in experimental models of rodent intracerebral hemorrhage.

T-lymphocytes promote cerebral inflammation, thus aggravating neuronal injury after stroke. Fingolimod, a sphingosine 1-phosphate receptor analog, prevents the egress of lymphocytes from primary and secondary lymphoid organs. Based on these findings, we hypothesized fingolimod treatment would reduce the number of T-lymphocytes migrating into the brain, thereby ameliorating cerebral inflammation following experimental intracerebral hemorrhage (ICH). We investigated the effects of fingolimod in two well-established murine models of ICH, implementing intrastriatal infusions of either bacterial collagenase (cICH) or autologous blood (bICH). Furthermore, we tested the long term neurological improvements by Fingolimod in a collagenase-induced rat model of ICH. Fingolimod, in contrast to vehicle administration alone, improved neurological functions and reduced brain edema at 24 and 72 h following experimental ICH in CD-1 mice (n=103; p<0.05). Significantly fewer lymphocytes were found in blood and brain samples of treated animals when compared to the vehicle group (p<0.05). Moreover, fingolimod treatment significantly reduced the expression of intercellular adhesion molecule-1 (ICAM-1), interferon-γ (INF-γ), and interleukin-17 (IL-17) in the mouse brain at 72 h post-cICH (p<0.05 compared to vehicle). Long-term neurocognitive performance and histopathological analysis were evaluated in Sprague-Dawley rats between 8 and 10 weeks post-cICH (n=28). Treated rats showed reduced spatial and motor learning deficits, along with significantly reduced brain atrophy and neuronal cell loss within the basal ganglia (p<0.05 compared to vehicle). We conclude that fingolimod treatment ameliorated cerebral inflammation, at least to some extent, by reducing the availability and subsequent brain infiltration of T-lymphocytes, which improved the short and long-term sequelae after experimental ICH in rodents.

Delayed hyperbaric oxygen therapy induces cell proliferation through stabilization of cAMP responsive element binding protein in the rat model of MCAo-induced ischemic brain injury.

Treatments that could extend the therapeutic window of opportunity for stroke patients are urgently needed. Early administration of hyperbaric oxygen therapy (HBOT) has been proven neuroprotective in the middle cerebral artery occlusion (MCAo) in rodents. Our aim was to determine: 1) whether delayed HBOT after permanent MCAo (pMCAo) can still convey neuroprotection and restorative cell proliferation, and 2) whether these beneficial effects rely on HBO-induced activation of protein phosphatase-1γ (PP1-γ) leading to a decreased phosphorylation and ubiquitination of CREB and hence its stabilization. The experiments were performed in one hundred thirty-two male Sprague-Dawley rats with the body weight ranging from 240 to 270 g. Permanent MCAo was induced with the intraluminal filament occluding the right middle cerebral artery (MCA). In the first experiment, HBOT (2.5 ATA, 1h daily for 10 days) was started 48 h after pMCAo. Neurobehavioral deficits and infarct size as well as cyclic AMP response element-binding protein (CREB) expression and BrdU-DAB staining in the hippocampus and the peri-infarct region were evaluated on day 14 and day 28 post-MCAo. In the second experiment, HBOT (2.5 ATA, 1h) was started 3h after pMCAo. The effects of CREB siRNA or PP1-γ siRNA on HBO-induced infarct size alterations and target protein expression were studied. HBOT started with 48 h delay reduced infarct size, ameliorated neurobehavioral deficits and increased protein expression of CREB, resulting in increased cell proliferations in the hippocampus and peri-infarct region, on day 14 and day 28 post-MCAo. In the acute experiment pMCAo resulted in cerebral infarction and functional deterioration and reduced brain expression of PP1-γ, which led to increased phosphorylation and ubiquitination of CREB 24h after MCAo. However HBOT administered 3h after ischemia reversed these molecular events and resulted in CREB stabilization, infarct size reduction and neurobehavioral improvement. Gene silencing with CREB siRNA or PP1-γ siRNA reduced acute beneficial effects of HBO. In conclusion, delayed daily HBOT presented as potent neuroprotectant in pMCAo rats, increased CREB expression and signaling activity, and bolstered regenerative type cell proliferation in the injured brain. As shown in the acute experiment these effects of HBO were likely to be mediated by reducing ubiquitin-dependent CREB degradation owing to HBO-induced activation of PP1γ.

Modeling intracerebral hemorrhage in mice: injection of autologous blood or bacterial collagenase.

Spontaneous intracerebral hemorrhage (ICH) defines a potentially life-threatening neurological malady that accounts for 10-15% of all stroke-related hospitalizations and for which no effective treatments are available to date(1,2). Because of the heterogeneity of ICH in humans, various preclinical models are needed to thoroughly explore prospective therapeutic strategies(3). Experimental ICH is commonly induced in rodents by intraparenchymal injection of either autologous blood or bacterial collagenase(4). The appropriate model is selected based on the pathophysiology of hemorrhage induction and injury progression. The blood injection model mimics a rapidly progressing hemorrhage. Alternatively, bacterial collagenase enzymatically disrupts the basal lamina of brain capillaries, causing an active bleed that generally evolves over several hours(5). Resultant perihematomal edema and neurofunctional deficits can be quantified from both models. In this study, we described and evaluated a modified double injection model of autologous whole blood(6) as well as an ICH injection model of bacterial collagenase(7), both of which target the basal ganglia (corpus striatum) of male CD-1 mice. We assessed neurofunctional deficits and brain edema at 24 and 72 hr after ICH induction. Intrastriatal injection of autologous blood (30 µl) or bacterial collagenase (0.075U) caused reproducible neurofunctional deficits in mice and significantly increased brain edema at 24 and 72 hr after surgery (p<0.05). In conclusion, both models yield consistent hemorrhagic infarcts and represent basic methods for preclinical ICH research.

PDGFR-α inhibition preserves blood-brain barrier after intracerebral hemorrhage.

OBJECTIVE: Perihematomal edema results from disruption of the blood-brain barrier (BBB) by key mediators, such as thrombin, following intracerebral hemorrhage (ICH). Platelet-derived growth factor receptor alpha (PDGFR-α), a tyrosine kinase receptor, was found in previous studies to play a role in orchestrating BBB impairment. In the present study, we investigated the role of PDGFR-α following ICH-induced brain injury in mice, specifically investigating its effect on BBB disruption. METHODS: Brain injury was induced by autologous arterial blood (30 µl) or thrombin (5 U) injection into mice brains. A PDGFR antagonist (Gleevec) or agonist (PDGF-AA) was administered following ICH. PDGF-AA was injected with a thrombin inhibitor, hirudin, in ICH mice. Thrombin-injected mice were given Gleevec or PDGF-AA neutralizing antibody. A p38 mitogen-activated protein kinase (MAPK) inhibitor, SB203580, was delivered with PDGF-AA in naïve animals. Postassessment included neurological function tests, brain edema measurement, Evans blue extravasation, immunoprecipitation, western blot, and immunohistology assay. RESULTS: PDGFR-α suppression prevented neurological deficits, brain edema, and Evans blue extravasation at 24 to 72 hours following ICH. PDGFR-α activation led to BBB impairment and this was reversed by SB203580 in naïve mice. Thrombin inhibition suppressed PDGFR-α activation and exogenous PDGF-AA increased PDGFR-α activation, regardless of thrombin inhibition. Animals receiving a PDGF-AA-neutralizing antibody or Gleevec showed minimized thrombin injection-induced BBB impairment. INTERPRETATION: PDGFR-α signaling may contribute to BBB impairment via p38 MAPK-mediated matrix metalloproteinase (MMP) activation/expression following ICH, and thrombin may be the key upstream orchestrator. The therapeutic interventions targeting the PDGFR-α signaling may be a novel strategy to prevent thrombin-induced BBB impairment following ICH.

Capsaicin pre-treatment provides neurovascular protection against neonatal hypoxic-ischemic brain injury in rats.

Capsaicin, a transient receptor potential vanilloid 1 (TRPV1) agonist, has recently been shown to provide neuroprotection against brain injury in experimental adult models of cerebral ischemia. Accordingly, in this study, we investigated the way in which capsaicin-mediated TRPV1 modulation could attenuate damage in an experimental hypoxic-ischemic (HI) neonatal brain injury model. The Rice-Vannucci method was used in 10-day-old rat pups by performing unilateral carotid artery ligation followed by 2 h of hypoxia (8% O2 at 37°C). Capsaicin was administered intraperitoneally (0.2 mg/kg or 2.0 mg/kg) at 3 h pre-HI or 1 h post-HI. Post assessment included measurement of infarction volume at 24 and 72 h in addition to an assessment of the vascular dynamics of the middle cerebral artery (MCA) at 6 h post-HI. The results indicated that pre-treatment with capsaicin reduced infarction volume significantly with either low-dose or high-dose treatment. Pre-treatment also improved myogenic tone and decreased apoptotic changes in the distal MCA. We concluded that capsaicin pre-treatment may provide neurovascular protection against neonatal HI.

Beneficial effect of hyperbaric oxygenation after neonatal germinal matrix hemorrhage.

BACKGROUND: Germinal matrix hemorrhage (GMH) is a potentially devastating neurological disease of very low birth weight premature infants. This leads to post-hemorrhagic hydrocephalus, cerebral palsy, and mental retardation. Hyperbaric oxygen (HBO) treatment is a broad neuroprotectant after brain injury. This study investigated the therapeutic effect of HBO after neonatal GMH. METHODS: Neonatal rats underwent stereotaxic infusion of clostridial collagenase into the right germinal matrix (anterior caudate) brain region. Cognitive function was assessed at 3 weeks, and then sensorimotor, cerebral, cardiac, and splenic growths were measured 1 week thereafter. RESULTS: Hyperbaric oxygen (HBO) treatment markedly improved upon the mental retardation and cerebral palsy outcome measurements in rats at the juvenile developmental stage. The administration of HBO early after neonatal GMH also normalized brain atrophy, splenomegaly, and cardiac hypertrophy 1 month after injury. CONCLUSION: This study supports the role of hyperbaric oxygen (HBO) treatment in the early period after neonatal GMH. HBO is an effective strategy to help protect the infant's brain from the post-hemorrhagic consequences of brain atrophy, mental retardation, and cerebral palsy. Further studies are necessary to determine the mechanistic basis of these neuroprotective effects.