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1.
Behav Brain Res ; 303: 44-52, 2016 Apr 15.
Article in English | MEDLINE | ID: mdl-26784560

ABSTRACT

Evidence from experimental animal studies convincingly argues for a role of pro-inflammatory cytokines due to surgical trauma in causing postoperative cognitive dysfunction. However, other studies have shown exposure to 2-4h of isoflurane anesthetic without surgical trauma can also impair cognitive function. We aimed to determine cytokine changes over time following isoflurane exposure in the presence and absence of surgery and examine subsequent cognitive function. Male rats were exposed to isoflurane (1.8%, 4h) with or without laparotomy or control conditions and tested in a contextual fear conditioning paradigm 8 days later. On day 9 rats were perfused, serum and hippocampal samples were collected and 24 cytokines were analysed. Groups of rats exposed as above were killed 6 or 48h after isoflurane exposure to examine early cytokine changes. Isoflurane exposure resulted in significantly less freezing in the contextual fear conditioning test (F(2,31)=6.11, P=0.006) and addition of laparotomy caused no further deficits (P>0.05). At 6h post isoflurane exposure an immunosuppressive response was observed in the serum while hippocampal cytokines were largely unchanged. These finding suggest isoflurane alone causes inflammatory changes and cognitive deficits. The addition of a laparotomy had a negligible effect. Early after isoflurane exposure changes in serum and hippocampal cytokines were divergent but by 9 days were aligned. At this time cytokines associated with memory deficits and brain injury processes were significantly elevated in serum and brain.


Subject(s)
Cytokines/metabolism , Hippocampus/drug effects , Isoflurane/adverse effects , Laparotomy/adverse effects , Memory Disorders/chemically induced , Animals , Conditioning, Classical/drug effects , Fear/drug effects , Hippocampus/metabolism , Male , Memory Disorders/metabolism , Rats , Rats, Sprague-Dawley
2.
J Alzheimers Dis ; 44(3): 995-1005, 2015.
Article in English | MEDLINE | ID: mdl-25380590

ABSTRACT

Post-operative cognitive dysfunction (POCD) predominantly affects the elderly who suffer memory and concentration deficits after anesthesia and surgery. Animal studies have demonstrated anesthetic alone may contribute to POCD but results are variable and little is known about common anesthetics other than isoflurane. The present study investigated dose-dependence of desflurane anesthesia in young adult and aged rats. We hypothesize higher concentrations of desflurane will result in memory impairment in the water maze and that impairment will be worse in aged rats. Effects of anesthesia (1 or 1.5 MAC, 4 h) desflurane, or sham exposure on cognition were investigated in young adult (3 months) and aged (20-24 months) rats at 1, 4, and 12 weeks post-exposure. The Morris water maze was used to assess acquisition and retention of spatial reference memory. Latency to find the hidden platform and swimming speed were compared between treatments. Aged rats showed significant impairment in task acquisition after exposure to 1.5 MAC, but not 1.0 MAC desflurane anesthetic when tested 1 week following exposure. Latency to find the platform and distance travelled were significantly longer in aged rats given 1.5 MAC desflurane (latency: F(1,108) = 19.71, p < 0.0001; distance: F(1,108) = 5.79, p = 0.018). Deficits were not long-lasting and were no longer present at 4 or 12 weeks. In contrast, young adult rats performed equally as well as sham-exposed control rats irrespective of desflurane dose. This study showed the effects of desflurane on learning and memory in the water maze are age and dose dependent and are brief in duration.


Subject(s)
Aging , Anesthetics, Inhalation/adverse effects , Isoflurane/analogs & derivatives , Memory Disorders/chemically induced , Age Factors , Animals , Desflurane , Dose-Response Relationship, Drug , Isoflurane/adverse effects , Male , Maze Learning/drug effects , Rats , Rats, Sprague-Dawley , Time Factors
3.
PLoS One ; 9(4): e95762, 2014.
Article in English | MEDLINE | ID: mdl-24752645

ABSTRACT

BACKGROUND: In this study, the neuroprotective effect of a novel nonpeptide AT2R agonist, C21, was examined in a conscious model of stroke to verify a class effect of AT2R agonists as neuroprotective agents. METHODS AND RESULTS: Spontaneously hypertensive rats (SHR) were pre-treated for 5 days prior to stroke with C21 alone or in combination with the AT2R antagonist PD123319. In a separate series of experiments C21 was administered in a series of 4 doses commencing 6 hours after stroke. A focal reperfusion model of ischemia was induced in conscious SHR by administering endothelin-1 to the middle cerebral artery (MCA). Motor coordination was assessed at 1 and 3 days after stroke and post mortem analyses of infarct volumes, microglia activation and neuronal survival were performed at 72 hours post MCA occlusion. When given prior to stroke, C21 dose dependently decreased infarct volume, which is consistent with the behavioural findings illustrating an improvement in motor deficit. During the pre-treatment protocol C21 was shown to enhance microglia activation, which are likely to be evoking protection by releasing brain derived neurotrophic factor. When drug administration was delayed until 6 hours after stroke, C21 still reduced brain injury. CONCLUSION: These results indicate that centrally administered C21 confers neuroprotection against stroke damage. This benefit is likely to involve various mechanisms, including microglial activation of endogenous repair and enhanced cerebroperfusion. Thus, we have confirmed the neuroprotective effect of AT2R stimulation using a nonpeptide compound which highlights the clinical potential of the AT2R agonists for future development.


Subject(s)
Hypertension/drug therapy , Neuroprotective Agents/therapeutic use , Receptor, Angiotensin, Type 2/agonists , Receptor, Angiotensin, Type 2/metabolism , Animals , Endothelin-1/pharmacology , Male , Rats , Reperfusion Injury/chemically induced , Reperfusion Injury/drug therapy
5.
Hypertension ; 60(6): 1531-7, 2012 Dec.
Article in English | MEDLINE | ID: mdl-23090772

ABSTRACT

We have demonstrated previously that pretreatment with an angiotensin II type 2 receptor (AT(2)R) agonist is neuroprotective against a subsequent stroke independent of any changes in blood pressure. Therefore, in the current study, we have examined the potential neuroprotective effect of AT(2)R stimulation initiated after stroke induction to mimic the clinical setting. Intracerebroventricular administration of the AT(2)R agonist CGP42112 was commenced 6 hours after an ischemic stroke had been induced in conscious spontaneously hypertensive rats. CGP42112 given over 4 doses in the same rats (3 µg/kg per dose centrally) at 6, 24, 48, and 72 hours after stroke induction reduced total infarct volume (32 ± 13 mm(3) versus vehicle, 170 ± 49 mm(3); P<0.05) and improved motor function. Furthermore, we have demonstrated that AT(2)R stimulation after stroke increased neuronal survival, decreased apoptosis, and caused an increase in the number of activated microglia in the core region of damage. The effects of CGP42112 were partially reversed with the coadministration of an AT(2)R antagonist, PD123319. Thus, the current study has shown for the first time that delayed central AT(2)R stimulation after a cerebral incident is neuroprotective in a conscious rat model of stroke.


Subject(s)
Brain Ischemia/drug therapy , Neuroprotective Agents/therapeutic use , Oligopeptides/therapeutic use , Receptor, Angiotensin, Type 2/agonists , Stroke/drug therapy , Animals , Apoptosis/drug effects , Blood Pressure/drug effects , Cell Survival/drug effects , Male , Motor Activity/drug effects , Neuroprotective Agents/pharmacology , Oligopeptides/pharmacology , Rats , Rats, Inbred SHR , Recovery of Function/drug effects
6.
Anesthesiology ; 117(5): 1091-101, 2012 Nov.
Article in English | MEDLINE | ID: mdl-22929734

ABSTRACT

BACKGROUND: Sevoflurane has been found to increase apoptosis and pathologic markers associated with Alzheimer disease, provoking concern over their potential contribution to postoperative cognitive dysfunction. METHODS: The effects of anesthesia with 1 minimum alveolar concentration of sevoflurane for 4 h or sham exposure on cognition were investigated in young adult and aged (20-24 months) rats at 1, 4, and 12 weeks postexposure. Spatial reference memory acquisition and retention were tested in the Morris water maze task. Latency to locate the hidden platform and swim speed were determined and compared between treatments. RESULTS: Sevoflurane anesthesia significantly reduced latency to find the hidden platform in both young adult (n = 10 per treatment, P < 0.0001) and aged rats (n = 7 per treatment, P < 0.0001) when tested 1 week after exposure. In young rats only, this improved acquisition learning was maintained at 4 (P = 0.003) but not at 12 weeks postexposure (P = 0.061). There were no differences in swim speed or in open field exploration between groups (no confounding effects of stress or locomotion). Retention memory measured using probe trials was not affected by exposure to sevoflurane in young adult or aged rats. CONCLUSION: Sevoflurane anesthesia did not impair acquisition learning and retention memory in young adult or aged rats.


Subject(s)
Anesthesia, Inhalation , Maze Learning/drug effects , Memory/drug effects , Methyl Ethers/pharmacology , Age Factors , Anesthesia, Inhalation/methods , Animals , Learning/drug effects , Learning/physiology , Male , Maze Learning/physiology , Memory/physiology , Random Allocation , Rats , Rats, Sprague-Dawley , Sevoflurane
7.
Exp Neurol ; 235(1): 326-35, 2012 May.
Article in English | MEDLINE | ID: mdl-22417925

ABSTRACT

Ubiquitin-modification of proteins by E3 ubiquitin ligases is an important post-translational mechanism implicated in neuronal survival and injury following cerebral ischemia. However, of the 500 or so E3s thought to be present in mammalian cells, very few specific E3s have been identified and associated with brain ischemia. Here, we demonstrate endogenous induction of HECT-type E3 ligases of the Nedd4 family and their adaptor Nedd4-family interacting protein 1 (Ndfip1) following transient focal cerebral ischemia in rats. Ndfip1 is upregulated in surviving cortical neurons and its neuroprotective activity is correlated with Nedd4-2 upregulation, but not two other Nedd4 family members examined (Nedd4-1 and Itch). Immunoprecipitation assays confirmed biochemical binding of Ndfip1 with Nedd4-2 in the brain, with or without ischemic stroke, indicating their endogenous interaction. While Ndfip1 and Itch have been previously shown to interact outside of the nervous system, ischemic induction of Itch in the present study was associated with cellular survival independent of Ndfip1. Together, these findings demonstrate specific and differential regulation of Nedd4 family E3 ligases under ischemic conditions, and identify two E3 ligases and their adaptor that potentially regulate ubiquitination in ischemic stroke to provide neuroprotection.


Subject(s)
Brain Ischemia/metabolism , Carrier Proteins/metabolism , Cerebral Cortex/metabolism , Endosomal Sorting Complexes Required for Transport/metabolism , Membrane Proteins/metabolism , Neurons/metabolism , Stroke/metabolism , Ubiquitin-Protein Ligases/metabolism , Animals , Apoptosis/physiology , Brain Ischemia/genetics , Carrier Proteins/genetics , Endosomal Sorting Complexes Required for Transport/genetics , Male , Membrane Proteins/genetics , Nedd4 Ubiquitin Protein Ligases , Rats , Rats, Wistar , Stroke/genetics , Ubiquitin-Protein Ligases/genetics , Up-Regulation
8.
Eur J Anaesthesiol ; 29(5): 239-45, 2012 May.
Article in English | MEDLINE | ID: mdl-22343609

ABSTRACT

BACKGROUND: Postoperative cognitive dysfunction has been reported in young, middle-aged and elderly patients with greater incidence with increasing age. Neurocognitive deficits are associated with anaesthetic exposure in aged rodents but in younger adult animals the findings are inconsistent and in middle-aged animals they are unknown. We aimed to compare the effects of moderate duration isoflurane anaesthesia in 100% oxygen on Morris water maze performance in young adult and middle-aged rats. We hypothesised that isoflurane would have greater effects on learning and memory in middle-aged compared with young rats. MATERIALS AND METHODS: Young adult (3 months, n = 25) and middle-aged (12 months, n = 20) male Sprague Dawley rats were assigned randomly to isoflurane exposure (1 minimum alveolar concentration, 4 h) or control conditions. Spatial learning (acquisition phase) and memory (probe trial) were tested in the Morris water maze 1 week after exposure. Middle-aged rats were retested in the probe trial 4 weeks after exposure for long-term memory retention. Latency to locate the hidden platform and time spent in the platform quadrant were compared between ages and treatments. RESULTS: Isoflurane did not affect acquisition of the water maze task in either age group. Isoflurane exposure induced a significant deficit in memory retention in young rats, but not middle-aged rats, in the probe trial 24 h after acquisition. Irrespective of treatment, middle-aged rats took longer to acquire the task than young rats. Four weeks after exposure, isoflurane-treated middle-aged rats showed no preference for target location, compared with sham-exposed rats which retained memory for previous platform location. CONCLUSION: Isoflurane exposure impaired retention memory for platform location 1 week after exposure in young adult rats and resulted in a delayed although weak impairment at 4 weeks in middle-aged rats. The great variation in reported effects of isoflurane suggests a complex effect of this agent on memory which warrants further investigation.


Subject(s)
Anesthetics, Inhalation/toxicity , Behavior, Animal/drug effects , Cognition Disorders/chemically induced , Cognition/drug effects , Isoflurane/toxicity , Age Factors , Animals , Cognition Disorders/physiopathology , Cognition Disorders/psychology , Learning/drug effects , Male , Memory/drug effects , Motor Activity/drug effects , Rats , Rats, Sprague-Dawley , Reaction Time
9.
J Cell Biol ; 196(1): 29-36, 2012 Jan 09.
Article in English | MEDLINE | ID: mdl-22213801

ABSTRACT

PTEN (phosphatase and tensin homologue deleted on chromosome TEN) is the major negative regulator of phosphatidylinositol 3-kinase signaling and has cell-specific functions including tumor suppression. Nuclear localization of PTEN is vital for tumor suppression; however, outside of cancer, the molecular and physiological events driving PTEN nuclear entry are unknown. In this paper, we demonstrate that cytoplasmic Pten was translocated into the nuclei of neurons after cerebral ischemia in mice. Critically, this transport event was dependent on a surge in the Nedd4 family-interacting protein 1 (Ndfip1), as neurons in Ndfip1-deficient mice failed to import Pten. Ndfip1 binds to Pten, resulting in enhanced ubiquitination by Nedd4 E3 ubiquitin ligases. In vitro, Ndfip1 overexpression increased the rate of Pten nuclear import detected by photobleaching experiments, whereas Ndfip1(-/-) fibroblasts showed negligible transport rates. In vivo, Ndfip1 mutant mice suffered larger infarct sizes associated with suppressed phosphorylated Akt activation. Our findings provide the first physiological example of when and why transient shuttling of nuclear Pten occurs and how this process is critical for neuron survival.


Subject(s)
Brain Ischemia/metabolism , Carrier Proteins/physiology , Membrane Proteins/physiology , Neurons/physiology , PTEN Phosphohydrolase/metabolism , Animals , Brain Ischemia/pathology , Carrier Proteins/genetics , Carrier Proteins/metabolism , Cell Survival , Endosomal Sorting Complexes Required for Transport/physiology , Intercellular Signaling Peptides and Proteins , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mice , Mice, Inbred C57BL , Nedd4 Ubiquitin Protein Ligases , Photobleaching , Protein Transport , Ubiquitin-Protein Ligases/physiology , Ubiquitination
10.
PLoS One ; 6(11): e26826, 2011.
Article in English | MEDLINE | ID: mdl-22073202

ABSTRACT

Spinal cord trauma in the adult nervous system usually results in permanent loss of function below the injury level. The immature spinal cord has greater capacity for repair and can develop considerable functionality by adulthood. This study used the marsupial laboratory opossum Monodelphis domestica, which is born at a very early stage of neural development. Complete spinal cord transection was made in the lower-thoracic region of pups at postnatal-day 7 (P7) or P28, and the animals grew to adulthood. Injury at P7 resulted in a dense neuronal tissue bridge that connected the two ends of the cord; retrograde neuronal labelling indicated that supraspinal and propriospinal innervation spanned the injury site. This repair was associated with pronounced behavioural recovery, coordinated gait and an ability to use hindlimbs when swimming. Injury at P28 resulted in a cyst-like cavity encased in scar tissue forming at the injury site. Using retrograde labelling, no labelled brainstem or propriospinal neurons were found above the lesion, indicating that detectable neuronal connectivity had not spanned the injury site. However, these animals could use their hindlimbs to take weight-supporting steps but could not use their hindlimbs when swimming. White matter, demonstrated by Luxol Fast Blue staining, was present in the injury site of P7- but not P28-injured animals. Overall, these studies demonstrated that provided spinal injury occurs early in development, regrowth of supraspinal innervation is possible. This repair appears to lead to improved functional outcomes. At older ages, even without detectable axonal growth spanning the injury site, substantial development of locomotion was still possible. This outcome is discussed in conjunction with preliminary findings of differences in the local propriospinal circuits following spinal cord injury (demonstrated with fluororuby labelling), which may underlie the weight bearing locomotion observed in the apparent absence of axons bridging the lesion site in P28-injured Monodelphis.


Subject(s)
Animals, Newborn , Locomotion , Opossums/physiology , Spinal Cord Injuries/physiopathology , Animals , Gait , Swimming
11.
J Neurophysiol ; 105(4): 1482-94, 2011 Apr.
Article in English | MEDLINE | ID: mdl-21273307

ABSTRACT

Recurring waves of peri-infarct depolarizations (PIDs) propagate across gray matter in the hours and days following stroke, expanding the primary site of injury. Ischemic depolarization (termed anoxic depolarization or AD in live brain slices) is PID-like but immediately arises in the more metabolically compromised ischemic core. This causes dramatic neuronal and astrocyte swelling and dendritic beading with spine loss within minutes, resulting in acute cell death. AD is evoked in rodent neocortical slices by suppressing the Na(+)/K(+)-ATPase pump with either oxygen/glucose deprivation (OGD) or exposure to ouabain. The process driving AD and PIDs remains poorly understood. Here we show that dibucaine is a potent drug inhibiting AD because of its high binding affinity to the Na(+) channel. Field recording reveals that, when superfused with ouabain (5 min), neocortical slices pretreated with 1 µM dibucaine for 45 min display either no AD or delayed AD onset compared with untreated controls. If ouabain exposure is extended to 10 min, 1 µM dibucaine is still able to delay AD onset by ∼ 60%. Likewise, it delays OGD-evoked AD onset by ∼ 54% but does not depress action potentials (APs) or evoked orthodromic field potentials. Increasing dibucaine to 10 µM inhibits AP firing, gradually putting the slice into a stasis that inhibits AD onset but also renders the slice functionally quiescent. Two-photon microscopy reveals that 10 µM dibucaine pretreatment prevents or helps reverse ouabain-induced structural neuronal damage. Although the therapeutic range of dibucaine is quite narrow, dibucaine-like drugs could prove therapeutically useful in inhibiting PIDs and their resultant neuronal damage.


Subject(s)
Dibucaine/therapeutic use , Hypoxia-Ischemia, Brain/pathology , Hypoxia-Ischemia, Brain/prevention & control , Sodium Channel Blockers/therapeutic use , Stroke/complications , Action Potentials/drug effects , Animals , Astrocytes/pathology , Dendrites/pathology , Dibucaine/pharmacology , Enzyme Inhibitors/pharmacology , Female , Hypoxia-Ischemia, Brain/etiology , Male , Mice , Mice, Transgenic , Models, Animal , Neurons/pathology , Ouabain/pharmacology , Rats , Rats, Sprague-Dawley , Sodium Channel Blockers/pharmacology , Sodium-Potassium-Exchanging ATPase/antagonists & inhibitors
12.
PLoS One ; 5(8): e12021, 2010 Aug 09.
Article in English | MEDLINE | ID: mdl-20711496

ABSTRACT

Cellular mechanisms of secondary damage progression following spinal cord injury remain unclear. We have studied the extent of tissue damage from 15 min to 10 weeks after injury using morphological and biochemical estimates of lesion volume and surviving grey and white matter. This has been achieved by semi-quantitative immunocytochemical methods for a range of cellular markers, quantitative counts of white matter axonal profiles in semi-thin sections and semi-quantitative Western blot analysis, together with behavioural tests (BBB scores, ledged beam, random rung horizontal ladder and DigiGait analysis). We have developed a new computer-controlled electronic impactor based on a linear motor that allows specification of the precise nature, extent and timing of the impact. Initial (15 min) lesion volumes showed very low variance (1.92+/-0.23 mm3, mean+/-SD, n=5). Although substantial tissue clearance continued for weeks after injury, loss of grey matter was rapid and complete by 24 hours, whereas loss of white matter extended up to one week. No change was found between one and 10 weeks after injury for almost all morphological and biochemical estimates of lesion size or behavioural methods. These results suggest that previously reported apparent ongoing injury progression is likely to be due, to a large extent, to clearance of tissue damaged by the primary impact rather than continuing cell death. The low variance of the impactor and the comprehensive assessment methods described in this paper provide an improved basis on which the effects of potential treatment regimes for spinal cord injury can be assessed.


Subject(s)
Central Nervous System/pathology , Contusions/pathology , Spinal Cord Injuries/pathology , Animals , Axons/pathology , Behavior, Animal , Blotting, Western , Cell Count , Central Nervous System/metabolism , Contusions/metabolism , Disease Progression , Male , Nerve Fibers, Myelinated/pathology , Neurons/pathology , Rats , Rats, Sprague-Dawley , Spinal Cord Injuries/metabolism , Time Factors
13.
Stroke ; 40(4): 1482-9, 2009 Apr.
Article in English | MEDLINE | ID: mdl-19246705

ABSTRACT

BACKGROUND AND PURPOSE: The angiotensin II type 2 receptor (AT(2)R) is implicated to be neuroprotective in stroke, although this premise has not been directly tested. Therefore, we have examined the neuroprotective effect of AT(2)R stimulation after intracerebroventricular administration of AT(2)R agonist CGP42112 in a conscious rat model of stroke. METHODS: Spontaneously hypertensive rats were treated with either CGP42112 (0.1 to 10 ng/kg/min intracerebroventricularly) alone or in combination with the AT(2)R antagonist PD123319 (36 ng/kg/min intracerebroventricularly) beginning 5 days before stroke induction. A focal reperfusion model of stroke was induced in conscious spontaneously hypertensive rats by administering endothelin-1 to the middle cerebral artery through a surgically implanted cannula. Behavioral tests were used to assess the severity of neurological deficit as a result of the ischemic event. Cortical and striatal infarct volumes were measured 72 hours poststroke. RESULTS: Blood pressure was unaffected by treatments. CGP42112 dose-dependently reduced cortical infarct volume poststroke, and PD123319 abolished the neuroprotective effect of CGP42112. PD123319 had no effect on infarct volume alone. These results were consistent with the behavioral findings, indicating that CGP42112 reduced motor deficit on the ledged beam test at 72 hours poststroke and immunohistochemical analyses showing that CGP42112 increased neuronal survival and minimized the loss of AT(2)R expression in the infarcted region. CONCLUSIONS: Based on infarct, behavioral, and immunohistochemical data, these results indicate that centrally administered CGP42112 exhibits a neuroprotective effect, which was independent of blood pressure. Thus, for the first time, we have shown that central AT(2)R stimulation is neuroprotective in a conscious rat model of stroke.


Subject(s)
Neuroprotective Agents/pharmacology , Oligopeptides/pharmacology , Receptor, Angiotensin, Type 2/agonists , Receptor, Angiotensin, Type 2/metabolism , Stroke/drug therapy , Animals , Blood Pressure/drug effects , Cell Survival/drug effects , Cerebral Infarction/drug therapy , Cerebral Infarction/metabolism , Cerebral Infarction/pathology , Consciousness , Disease Models, Animal , Immunohistochemistry , Injections, Intraventricular , Male , Motor Activity/drug effects , Rats , Rats, Inbred SHR , Stroke/metabolism , Stroke/pathology , Superoxides/metabolism , Vasodilator Agents/pharmacology
14.
Pediatr Res ; 63(6): 620-4, 2008 Jun.
Article in English | MEDLINE | ID: mdl-18317402

ABSTRACT

Preconditioning with hypoxia and hypoxia-mimetic compounds cobalt chloride (CoCl2) and desferrioxamine (DFX) protects against hypoxic-ischemic (HI) injury in neonatal rat brain. We examined long-term functional and protective actions of preconditioning induced by hypoxia, CoCl(2) and DFX in a neonatal rat model of HI. Postnatal day six rat pups were exposed to preconditioning with hypoxia (8% oxygen) or injections of CoCl(2), DFX or saline vehicle and 24 h later rats underwent HI or sham surgery. Behavioral tests were performed and at the conclusion of experiments, brains removed for morphologic analyses. HI resulted in a large unilateral lesion in the ipsilateral hemisphere compared with sham control rats. All preconditioning treatments significantly reduced the total lesion volume. Behavioral deficits were observed in HI rats compared with sham controls. The reduction in forelimb grasping strength in HI rats was attenuated by preconditioning with hypoxia, CoCl(2) and DFX. HI increased the number of foot faults in a grid-walking test and resulted in forelimb asymmetry in the cylinder test. Only preconditioning with hypoxia reversed all three functional deficits after HI. These findings indicate that preconditioning, especially when induced by hypoxia, has the potential to minimize the morphologic and functional effects of neonatal HI injury.


Subject(s)
Brain/drug effects , Cobalt/pharmacology , Deferoxamine/pharmacology , Hypoxia-Ischemia, Brain/prevention & control , Hypoxia/physiopathology , Neuroprotective Agents/pharmacology , Animals , Animals, Newborn , Behavior, Animal/drug effects , Brain/pathology , Brain/physiopathology , Cobalt/therapeutic use , Deferoxamine/therapeutic use , Disease Models, Animal , Female , Hypoxia/pathology , Hypoxia-Ischemia, Brain/pathology , Hypoxia-Ischemia, Brain/physiopathology , Male , Motor Skills/drug effects , Neuroprotective Agents/therapeutic use , Rats , Rats, Sprague-Dawley , Time Factors
15.
Eur J Pharmacol ; 584(1): 100-10, 2008 Apr 14.
Article in English | MEDLINE | ID: mdl-18316070

ABSTRACT

Prognostic models are used to predict outcome in stroke patients and to stratify treatment groups in clinical trials. No one has previously attempted to use such models in stroke recovery studies in animals. We have now shown the predictive value of assigning stroke severity ratings, based on behaviours displayed in conscious rats during infusion of endothelin-1 to constrict the middle cerebral artery, on neurological and histological outcomes. The validity of prior stratification of treatment groups according to stroke ratings was tested by assessment of the protective potential of synthetic flavonol, 3',4'-dihydroxyflavonol (DiOHF). Neurological deficits and performance on the sticky label test were evaluated before and at 24, 48 and 72 h post-stroke. Histopathology was assessed at 72 h. Positive correlations between stroke ratings and neurological deficit scores were found at 24 (r=0.58, P<0.001), 48 (r=0.53, P<0.001) and 72 (r=0.56, P<0.001) h post-stroke, with more severe strokes associated with worse deficit scores. Similar correlations were observed with the sticky label test. Higher stroke ratings also correlated with greater infarct volumes (total infarct volume: r=0.74, P<0.0001). Treatment with DiOHF (10 mg/kg i.v. given 3, 24 and 48 h post-stroke) significantly reduced infarct volume and restored neurological function in rats with modest stroke ratings (P<0.01), but not in rats with high stroke ratings. These results suggest that stroke ratings, based on behavioural assessment as the stroke develops, reliably predict histopathological and functional outcomes and allow stratification of treatment groups. DiOHF given after stroke improves outcomes in moderate strokes, and therefore has cytoprotective potential.


Subject(s)
Behavior, Animal/drug effects , Flavonols/pharmacology , Infarction, Middle Cerebral Artery/drug therapy , Neuroprotective Agents/pharmacology , Animals , Brain Mapping , Consciousness , Cytoprotection , Disease Models, Animal , Endothelin-1 , Flavonols/therapeutic use , Infarction, Middle Cerebral Artery/chemically induced , Infarction, Middle Cerebral Artery/pathology , Infarction, Middle Cerebral Artery/physiopathology , Male , Neuroprotective Agents/therapeutic use , Prognosis , Rats , Rats, Wistar , Reproducibility of Results , Severity of Illness Index , Time Factors
16.
J Pharm Pharmacol ; 60(2): 171-8, 2008 Feb.
Article in English | MEDLINE | ID: mdl-18237464

ABSTRACT

The plasma pharmacokinetics and brain uptake of the novel neuroprotective agent AM-36 (1-(2-(4-chlorophenyl)-2-hydroxy)ethyl-4-(3,5-bis-(1,1dimethylethyl)-4-hydroxyphenyl) methylpiperazine) were assessed over 72 h following i.v. administration to male Sprague-Dawley rats. At nominal i.v. doses of 0.2, 1 and 3mg kg(-1), AM-36 exhibited an extremely large volume of distribution (18.2-24.6 L kg(-1)) and a long terminal elimination half-life, ranging from 25.2 to 37.7 h. Over this dose range, AM-36 exhibited linear pharmacokinetics, with no apparent change in clearance, volume of distribution or dose-normalised area under the plasma concentration - time curve. AM-36 was very highly bound to plasma proteins (> 99.6%); however, this did not appear to affect the ability of AM-36 to permeate the blood-brain barrier. Following a single i.v. dose of AM-36 at 3mg kg(-1) to rats, brain concentrations were detected for up to 72 h, and the brain-to-plasma ratios were high at all time points (ranging from 8.2 at 5 min post-dose to 0.9 at 72 h post-dose). The very high brain uptake of AM-36 supports previous in-vivo efficacy studies demonstrating the neuroprotective effects of this compound when administered to rats with middle cerebral artery occlusion.


Subject(s)
Blood-Brain Barrier/metabolism , Brain/metabolism , Neuroprotective Agents/pharmacokinetics , Piperazines/pharmacokinetics , Animals , Area Under Curve , Dose-Response Relationship, Drug , Half-Life , Injections, Intravenous , Male , Neuroprotective Agents/administration & dosage , Piperazines/administration & dosage , Protein Binding , Rats , Rats, Sprague-Dawley , Time Factors , Tissue Distribution
17.
J Cereb Blood Flow Metab ; 27(1): 100-14, 2007 Jan.
Article in English | MEDLINE | ID: mdl-16736051

ABSTRACT

Accumulation of neutrophils in brain after transient focal stroke remains controversial with some studies showing neutrophils to be deleterious, whereas others suggest neutrophils do not contribute to ischemic injury. Myeloperoxidase (MPO) has been used extensively as a marker for quantifying neutrophil accumulation, but is an indirect method and does not detect neutrophils alone. To elucidate the interaction of macrophages in the neutrophil inflammatory response, we conducted double-label immunofluorescence in brain sections at 0, 1, 2, 3, 7, and 15 days after ischemia. Each of these results was obtained from the same animal to determine correlations between neutrophil infiltration and ischemic damage. It was found that MPO activity increased up to 3 days after cerebral ischemia. Dual-staining revealed that macrophages engulf neutrophils in the brain and that this engulfment of neutrophils increased with time, with 50% of neutrophils in the brain engulfed at 3 days and approximately 85% at 15 days (N=5, P<0.05). Interestingly, at 7 days the amount of dual-staining was decreased to 20% (N=5, P<0.05). Neutrophil infiltration was positively correlated with ischemic damage in both the cortex and striatum (r(2)=0.86 and 0.80, respectively, P<0.01). The results of this study indicate that the MPO from neutrophils phagocytized by macrophages may continue to contribute to the overall MPO activity, and that previous assessments that have utilized this marker to measure neutrophil accumulation may have mis-calculated the number of neutrophils within the ischemic territory and hence their contribution to the evolution of the infarct at later time points. Thus any biphasic infiltration of neutrophils may have been masked by the accumulation of macrophages.


Subject(s)
Brain Ischemia/chemically induced , Brain Ischemia/pathology , Endothelin-1 , Inflammation/pathology , Neutrophil Infiltration/physiology , Peroxidase/metabolism , Animals , Fluorescent Antibody Technique , Immunohistochemistry , Infarction, Middle Cerebral Artery/pathology , Macrophages/physiology , Male , Microscopy, Confocal , Middle Cerebral Artery/physiology , Rats , Rats, Long-Evans , Stereotaxic Techniques
18.
Exp Neurol ; 204(1): 220-33, 2007 Mar.
Article in English | MEDLINE | ID: mdl-17188268

ABSTRACT

Cerebral inflammation and apoptotic cell death are two processes implicated in the progressive tissue damage that occurs following traumatic brain injury (TBI), and strategies to inhibit one or both of these pathways are being investigated as potential therapies for TBI patients. The tetracycline derivative minocycline was therapeutically effective in various models of central nervous system injury and disease, via mechanisms involving suppression of inflammation and apoptosis. We therefore investigated the effect of minocycline in TBI using a closed head injury model. Following TBI, mice were treated with minocycline or vehicle, and the effect on neurological outcome, lesion volume, inflammation and apoptosis was evaluated for up to 7 days. Our results show that while minocycline decreases lesion volume and improves neurological outcome at 1 day post-trauma, this response is not maintained at 4 days. The early beneficial effect is likely not due to anti-apoptotic mechanisms, as the density of apoptotic cells is not affected at either time-point. However, protection by minocycline is associated with a selective anti-inflammatory response, in that microglial activation and interleukin-1beta expression are reduced, while neutrophil infiltration and expression of multiple cytokines are not affected. These findings demonstrate that further studies on minocycline in TBI are necessary in order to consider it as a novel therapy for brain-injured patients.


Subject(s)
Apoptosis/drug effects , Brain Injuries/physiopathology , Microglia/drug effects , Minocycline/pharmacology , Neuroprotective Agents/pharmacology , Neutrophil Infiltration/drug effects , Animals , Brain/drug effects , Brain/pathology , Brain/physiopathology , Brain Injuries/metabolism , Brain Injuries/pathology , Cell Count , Cerebral Cortex/metabolism , Cerebral Cortex/pathology , Head Injuries, Closed/physiopathology , Interleukin-1beta/metabolism , Macrophages/pathology , Male , Mice , Mice, Inbred C57BL , Microglia/pathology , Motor Activity/drug effects , Nervous System/drug effects , Nervous System/physiopathology , Neutrophils/pathology , Time Factors
19.
Brain Res ; 1110(1): 13-22, 2006 Sep 19.
Article in English | MEDLINE | ID: mdl-16905121

ABSTRACT

Injury to axons and oligodendrocytes has been poorly characterized in most animal models of stroke, and hence has been difficult to target therapeutically. It is therefore necessary to characterize axonal and oligodendroglial injury in these models, in order to rationally design putative protective compounds that minimize this injury. This study aims to characterize injury to axons and oligodendrocytes in the endothelin-1 (ET-1) model of middle cerebral artery occlusion (MCAO) in conscious rats. Transient forebrain ischemia was induced in conscious adult male Long Evans rats by the perivascular microinjection of ET-1. Quantitative histopathology was performed on forebrain sections at 6, 24, 48 and 72 h after ET-1 administration, using ballistic light analyses and immunohistochemistry for amyloid precursor protein (APP), SMI32, and Tau-1. Ballistic light analyses of cortical and striatal lesions revealed that the infarct volume was maximal in these regions by 6 h. APP and SMI32 immunohistochemistry demonstrated that axonal injury was maximal by 6 h in this model; however, some injured axons appeared to maintain good structural integrity up to 72 h after insult. Density measurements for Tau-1-immunopositive oligodendrocytes were significantly elevated within the corpus callosum from 48 h, but reductions in total oligodendrocyte numbers were not apparent up 72 h after ET-1 injection. These results indicate that axonal and oligodendroglial injury should be investigated as potential targets for delayed therapeutic intervention after MCAO.


Subject(s)
Diffuse Axonal Injury/pathology , Endothelin-1 , Infarction, Middle Cerebral Artery/chemically induced , Oligodendroglia/pathology , Amyloid beta-Protein Precursor/metabolism , Animals , Antibodies, Monoclonal/metabolism , Brain/metabolism , Brain/pathology , Cell Count/methods , Diffuse Axonal Injury/etiology , Disease Models, Animal , Immunohistochemistry/methods , Infarction, Middle Cerebral Artery/pathology , Infarction, Middle Cerebral Artery/physiopathology , Male , Neurofilament Proteins/metabolism , Rats , Rats, Long-Evans , Time Factors
20.
Neurochem Int ; 48(6-7): 604-10, 2006.
Article in English | MEDLINE | ID: mdl-16530295

ABSTRACT

Excitatory amino acid transporters (EAATs) are responsible for homeostasis of extracellular L-glutamate, and the glial transporters are functionally dominant. EAAT expression or function is altered in acute and chronic neurological conditions, but little is known about the regulation of EAATs in reactive astroglia found in such neuropathologies. These studies examined the effects of the bacterial endotoxin lipopolysaccharide (LPS) on glial EAATs in vitro. The effects of LPS (1 microg/ml, 24-72 h) on EAAT activity and expression were examined in primary cultures of mouse astrocytes. [(3)H]D-aspartate uptake increased to 129% of control by 72 h treatment with LPS. Saturation analysis revealed that apparent K(m) was unchanged whilst V(max) was significantly increased to 172% of control by 72 h LPS treatment. Biotinylation and Western blotting indicated that cell-surface expression of GLT-1 was significantly elevated (146% control) by LPS treatment whereas GLAST expression was unchanged. Confocal analyses revealed that LPS treatment resulted in cytoskeletal changes and stellation of astrocytes, with rearrangement of F-actin (as shown by phalloidin labelling). Immunocytochemistry revealed clustering of GLAST, and increased expression and redistribution of GLT-1 to the cell-surface following treatment with LPS. Similar experiments were conducted in microglia, where LPS (50 ng/ml) was found to up-regulate expression of GLT-1 at 24 and 72 h in concert with cytoskeletal changes accompanying activation. These findings suggest an association of cytoskeletal changes in glia with EAAT activity, with the predominant adaptation involving up-regulation and redistribution of GLT-1.


Subject(s)
Excitatory Amino Acid Transporter 2/metabolism , Lipopolysaccharides/pharmacology , Neuroglia/drug effects , Actins/metabolism , Animals , Animals, Newborn , Aspartic Acid/metabolism , Astrocytes/drug effects , Astrocytes/ultrastructure , Biotinylation , Blotting, Western , Cells, Cultured , Cytoskeleton/drug effects , Cytoskeleton/metabolism , Cytoskeleton/ultrastructure , Excitatory Amino Acid Transporter 1/biosynthesis , Excitatory Amino Acid Transporter 2/biosynthesis , Immunohistochemistry , Mice , Mice, Inbred C57BL , Microglia/metabolism , Microscopy, Confocal , Neuroglia/cytology , Neuroglia/metabolism , Phenotype , Protein Transport , Up-Regulation
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