Structural changes in perineuronal nets and their perforating GABAergic synapses precede motor coordination recovery post stroke.

BACKGROUND: Stroke remains one of the leading causes of long-term disability worldwide, and the development of effective restorative therapies is hindered by an incomplete understanding of intrinsic brain recovery mechanisms. Growing evidence indicates that the brain extracellular matrix (ECM) has major implications for neuroplasticity. Here we explored how perineuronal nets (PNNs), the facet-like ECM layers surrounding fast-spiking interneurons, contribute to neurological recovery after focal cerebral ischemia in mice with and without induced stroke tolerance. METHODS: We investigated the structural remodeling of PNNs after stroke using 3D superresolution stimulated emission depletion (STED) and structured illumination (SR-SIM) microscopy. Superresolution imaging allowed for the precise reconstruction of PNN morphology using graphs, which are mathematical constructs designed for topological analysis. Focal cerebral ischemia was induced by transient occlusion of the middle cerebral artery (tMCAO). PNN-associated synapses and contacts with microglia/macrophages were quantified using high-resolution confocal microscopy. RESULTS: PNNs undergo transient structural changes after stroke allowing for the dynamic reorganization of GABAergic input to motor cortical L5 interneurons. The coherent remodeling of PNNs and their perforating inhibitory synapses precedes the recovery of motor coordination after stroke and depends on the severity of the ischemic injury. Morphological alterations in PNNs correlate with the increased surface of contact between activated microglia/macrophages and PNN-coated neurons. CONCLUSIONS: Our data indicate a novel mechanism of post stroke neuroplasticity involving the tripartite interaction between PNNs, synapses, and microglia/macrophages. We propose that prolonging PNN loosening during the post-acute period can extend the opening neuroplasticity window into the chronic stroke phase.

Shared Mechanisms of GABAergic and Opioidergic Transmission Regulate Corticolimbic Reward Systems and Cognitive Aspects of Motivational Behaviors.

The functional interplay between the corticolimbic GABAergic and opioidergic systems plays a crucial role in regulating the reward system and cognitive aspects of motivational behaviors leading to the development of addictive behaviors and disorders. This review provides a summary of the shared mechanisms of GABAergic and opioidergic transmission, which modulate the activity of dopaminergic neurons located in the ventral tegmental area (VTA), the central hub of the reward mechanisms. This review comprehensively covers the neuroanatomical and neurobiological aspects of corticolimbic inhibitory neurons that express opioid receptors, which act as modulators of corticolimbic GABAergic transmission. The presence of opioid and GABA receptors on the same neurons allows for the modulation of the activity of dopaminergic neurons in the ventral tegmental area, which plays a key role in the reward mechanisms of the brain. This colocalization of receptors and their immunochemical markers can provide a comprehensive understanding for clinicians and researchers, revealing the neuronal circuits that contribute to the reward system. Moreover, this review highlights the importance of GABAergic transmission-induced neuroplasticity under the modulation of opioid receptors. It discusses their interactive role in reinforcement learning, network oscillation, aversive behaviors, and local feedback or feedforward inhibitions in reward mechanisms. Understanding the shared mechanisms of these systems may lead to the development of new therapeutic approaches for addiction, reward-related disorders, and drug-induced cognitive impairment.

Long-term administration of bumetanide improve functional recovery after spinal cord injury in rats.

Ion disturbances are among the most remarkable deficits in spinal cord injury (SCI). GABA is an integral part of neural interaction. Action of the GABAA receptor depends on the amount of intracellular chloride. Homeostasis of chloride is controlled by two co-transporters, NKCC1 and KCC2. Previous studies revealed that NKCC1 are disturbed in SCI. In this study, NKCC1 is highly expressed in the epicenter of the lesioned spinal cord at 3 hours after induction of the lesion and reached the peak around 6 hours after SCI. Bumetanide (2 and 4 mg/day), as a specific NKCC1 inhibitor, was used at 3 hours post SCI for 28 days. The functional recovery outcomes were measured by the Basso-Beattie-Bresnahan (BBB) locomotor rating scale, ladder walking test, and hot plate test. The rats that received bumetanide 4 mg/day exhibited improved recovery of locomotor function, reduction of NKCC1 gene expression, and upregulation of GAP protein levels 28 days post SCI. Histological tissue evaluations confirmed bumetanide's neuroprotective and regenerative effects. This study provides novel evidence for the benefits of bumetanide in early administration after SCI.

Preconditioning exercise reduces hippocampal neuronal damage via increasing Klotho expression in ischemic rats.

Considerable amounts of oxidants are produced in cerebral ischemia, where oxidative stress plays a key role in neuronal damage after ischemia. Klotho, an anti-aging protein, alleviates oxidative stress by activating the transcription of an important antioxidant enzyme, manganese superoxide dismutase (MnSOD), in the nervous system. Thus, increased Klotho expression level could lead to a reduction in neuronal damages after brain ischemia via lowering oxidative stress. It is known that physical activity increases Klotho expressions. In this study, we assessed neuroprotective effects of preconditioning exercise in rats (treadmill running at a speed of 20 m/min,30 min/day, six days/week, for3 weeks) on hippocampal Klotho and MnSOD expression in the brain using an animal model of stroke, middle cerebral artery occlusion (MCAO). Our study revealed a reduction in hippocampal Klotho and MnSOD expression as well as CA1 neuronal activity in MCAO compared to the sham group. Exercise prevented the ischemia-induced decline in Klotho and MnSOD expression levels as well as CA1 neuronal activity in Exercise + MCAO compared to the MCAO group. Also, exercise significantly improved the neurological scores and reduced brain infarction area in Exercise + MCAO in comparison to MCAO group. There was a post-ischemia deficit in the working memory, as measured by spontaneous alternation percentage using Y-maze test, in MCAO compared to the sham group. The latter effect was not observed in the Exercise + MCAO group, which could be related to an increase in the antioxidant capacity as exhibited by Klotho and MnSOD up-regulation. The results were confirmed with a positive correlation between Klotho expression and MnSOD expression which allows proposing Klotho as a potential neuroprotective protein in ischemic stroke with respect to antioxidant defense. In general, the present study suggested that preconditioning exercise induced upregulation of Klotho and MnSOD, as well as attenuated the post-ischemic injuries. The upregulation of Klotho might be an underlying mechanism by which preconditioning exercise plays as a neuroprotective factor against post-ischemic neural injuries in ischemic rats.

N-Methyl-D-Aspartate Receptors Antagonist Prevents Secondary Ischemic Brain Injury Associated With Lipopolysaccharide-Induced Sepsis-Like State Presumably via Immunomodulatory Actions.

Infection is a major reason for poor stroke outcomes, and sepsis is a major cause of stroke-elated deaths. We herein examined whether NMDA receptor blockade, which was reported to exert anti-inflammatory actions, protects against the deleterious consequences of lipopolysaccharide (LPS)-induced sepsis-like state in adult male NMRI mice exposed to transient intraluminal middle cerebral artery occlusion (MCAO). At 24 h post-ischemia, vehicle or Escherichia coli LPS (2 or 4 mg/kg) was intraperitoneally administered, whereas 30 min later vehicle or ketamine (10 mg/kg), which is a non-competitive NMDA receptor antagonist, was intraperitoneally applied. Delivery of LPS at a dosage of 4 mg/kg induced a sepsis-like state characterized by a rectal temperature reduction by ∼4.0°C, increased neurological deficits in Clark score, cylinder and open-field tests, increased brain infarct volume and reduced neuronal survival in the previously ischemic tissue. Notably, additional treatment with ketamine (10 mg/kg) significantly attenuated the sepsis-associated rectal temperature reduction by ∼1.5°C, reduced neurological deficits, reduced infarct volume, and promoted neuronal survival. Ketamine alone did not influence infarct volume or neurological deficits. Real-time PCR data analysis showed that GFAP, CD86, CD206, IL-1ß, and IL-10 mRNA levels were significantly increased in ischemic brains of LPS-treated compared with vehicle-treated mice. Additional treatment with ketamine significantly decreased IL-1ß and IL-10, but not GFAP, CD86, and CD206 mRNA levels. Our data show that ketamine at a dose that on its own does not confer neuroprotection reverses the adverse effects of LPS-induced sepsis-like state post-ischemia, presumably via immunomodulatory actions.

Post-ischemic protein restriction induces sustained neuroprotection, neurological recovery, brain remodeling, and gut microbiota rebalancing.

BACKGROUND: Moderate dietary protein restriction confers neuroprotection when applied before ischemic stroke. How a moderately protein-reduced diet influences stroke recovery when administered after stroke, is a clinically relevant question. This question has not yet been investigated. METHODS: Male C57BL6/J mice were exposed to transient intraluminal middle cerebral artery occlusion. Immediately after the stroke, mice were randomized to two normocaloric diets: a moderately protein-reduced diet containing 8% protein (PRD) or normal diet containing 20% protein (ND). Post-stroke neurological deficits were evaluated by a comprehensive test battery. Antioxidant and neuroinflammatory responses in the brain and liver were evaluated by Western blot and RTqPCR. Stroke-induced brain injury, microvascular integrity, glial responses, and neuroplasticity were assessed by immunohistochemistry. Fecal microbiota analysis was performed using 16S ribosomal RNA amplicon sequencing. RESULTS: We show that PRD reduces brain infarct volume after three days and enhances neurological and, specifically, motor-coordination recovery over six weeks in stroke mice. The recovery-promoting effects of PRD were associated with increased antioxidant responses and reduced neuroinflammation. Histochemical studies revealed that PRD increased long-term neuronal survival, increased peri-infarct microvascular density, reduced microglia/macrophage accumulation, increased contralesional pyramidal tract plasticity, and reduced brain atrophy. Fecal microbiota analysis showed reduced bacterial richness and diversity in ischemic mice on ND starting at 7 dpi. PRD restored bacterial richness and diversity at these time points. CONCLUSION: Moderate dietary protein restriction initiated post-ischemic stroke induces neurological recovery, brain remodeling, and neuroplasticity in mice by mechanisms involving antiinflammation and, in the post-acute phase, commensal gut microbiota rebalancing.

Dopamine receptors in the anterior cingulate cortex implicate in nicotine enhanced morphine analgesia.

RATIONALE: It is well known that the anterior cingulate cortex (ACC) plays an important role in acute pain perception. OBJECTIVES: In the present study, we aimed to investigate the possible involvement of the ACC dopamine D1 and D2 receptors in nicotine plus morphine-induced analgesia. METHODS: The ACC's of adult male Wistar rats were bilaterally cannulated by stereotaxic instrument and the tail-flick test was used to measure the thermal pain threshold. RESULTS: The results indicated that subcutaneous (s.c.) injection of nicotine (0.3 mg/kg) potentiated the analgesic response of intraperitoneal (i.p.) administration of morphine (3 mg/kg). Systemic administration of the same doses of nicotine or morphine alone had no effect on tail-flick latency. Intra-ACC administration of apomorphine (0.3-0.9 µg/rat), the non-selective D1/D2 receptors agonist, plus ineffective doses of nicotine (0.1 mg/kg, s.c.) plus morphine (3 mg/kg, i.p) induced analgesia in rats. In addition, the analgesia induced with co-administration of nicotine and morphine was inhibited via intra-ACC administration of SCH23390 (0.5-1 µg/rat) or sulpiride (0.5-2 µg/rat), the selective antagonists of D1 or D2 receptors, respectively. The intra-ACC microinjection of the same doses of drugs alone had no effect on tail-flick latency. Cubic interpolation analysis also confirmed that activation or inactivation of the ACC D1 and D2 receptors by different doses of drugs can modulate the nicotine-morphine analgesic response. CONCLUSIONS: The findings suggest that the ACC has an important role in acute thermal pain perception and modulates the analgesia induced by nicotine plus morphine via dopaminergic receptors.

Hypocaloric Diet Initiated Post-Ischemia Provides Long-Term Neuroprotection and Promotes Peri-Infarct Brain Remodeling by Regulating Metabolic and Survival-Promoting Proteins.

Calorie restriction confers post-ischemic neuroprotection, when administered in a defined time window before ischemic stroke. How a hypocaloric diet influences stroke recovery when initiated after stroke has not been investigated. Male C57BL6/j mice were exposed to transient intraluminal middle cerebral artery occlusion. Immediately post-ischemia, mice were randomized to two groups receiving moderately hypocaloric (2286 kcal/kg food) or normocaloric (3518 kcal/kg) diets ad libitum. Animals were sacrificed at 3 or 56 days post-ischemia (dpi). Besides increased low density lipoprotein at 3 days and reduced alanine aminotransferase and increased urea at 56 days, no alterations of plasma markers were found in ischemic mice on hypocaloric diet. Body weight mildly decreased over 56 dpi by 7.4%. Hypocaloric diet reduced infarct volume in the acute stroke phase at 3 dpi and decreased brain atrophy, increased neuronal survival and brain capillary density in peri-infarct striatum and reduced motor coordination impairment in tight rope tests in the post-acute stroke phase over up to 56 dpi. The abundance of brain-derived neurotrophic factor, the NAD-dependent deacetylase and longevity protein sirtuin-1, the anti-oxidant glutathione peroxidase-3, and the ammonium detoxifier glutamine synthetase in the peri-infarct brain tissue was increased by hypocaloric diet. This study shows that a moderately hypocaloric diet that is initiated after stroke confers long-term neuroprotection and promotes peri-infarct brain remodeling.

Lipopolysaccharide-induced sepsis-like state compromises post-ischemic neurological recovery, brain tissue survival and remodeling via mechanisms involving microvascular thrombosis and brain T cell infiltration.

Sepsis predisposes for poor stroke outcome. This association suggests that sepsis disturbs post-ischemic tissue survival and brain remodeling. To elucidate this link, we herein exposed mice to 30 min intraluminal middle cerebral artery occlusion (MCAO) and induced a sepsis-like state at 72 h post-ischemia by intraperitoneal delivery of Escherichia coli lipopolysaccharide (LPS; three doses of 0.1 or 1 mg/kg, separated by 6 h), a major component of the bacterium's outer membrane. Neurological recovery, ischemic injury, brain remodeling and immune responses were evaluated over up to 56 days post-sepsis (dps) by behavioral tests, immunohistochemistry and flow cytometry. Delivery of 1 mg/kg but not 0.1 mg/kg LPS reduced rectal temperature over 48 h by up to 3.4 ± 3.1 °C, increased general and focal neurological deficits in the Clark score over 72 h and increased motor-coordination deficits in the tight rope test over up to 21 days. Notably, 1 mg/kg, but not 0.1 mg/kg LPS increased intercellular adhesion molecule-1 abundance on ischemic microvessels, increased microvascular thrombosis and increased patrolling monocyte and T cell infiltrates in ischemic brain tissue at 3 dps. Infarct volume was increased by 1 mg/kg, but not 0.1 mg/kg LPS at 3 dps (that is, 6 days post-MCAO), as was brain atrophy at 28 and 56 dps. Microglial activation in ischemic brain tissue, evaluated by morphology analysis of Iba-1 immunostainings, was transiently increased by 0.1 and 1 mg/kg LPS at 3 dps. Our data provide evidence that neurological recovery and brain remodeling are profoundly compromised in the ischemic brain post-sepsis as a consequence of cerebral thromboinflammation.

Expression analysis of hippocampal and amygdala CREB-BDNF signaling pathway in nicotine-induced reward under stress in rats.

Extensive research has shown that individuals are more sensitive to develop addiction and drug taking under stress state. The present study includes an expression analysis to identify the possible role of hippocampal and amygdala CREB (cAMP response element-binding protein) and BDNF (Brain-derived neurotrophic factor) activation in nicotine-induced conditioned place preference (CPP) under exposure to acute or sub-chronic stress. Using western-blot technique, CREB phosphorylation was shown to increase in the hippocampus and the amygdala following nicotine-induced CPP. The hippocampal level of BDNF was increased following nicotine administration and in the nicotine-treated animals exposed to acute stress. In animals exposed to acute stress, the amygdala ratios of the pCREB/CREB decreased, while pre-treatment of the animals with nicotine (0.1 mg/kg) decreased this ratio only in the hippocampus. Sub-chronic stress decreased the pCREB/CREB ratios in the hippocampus and the amygdala. Interestingly, sub-chronic stress-induced increase of nicotine reward only decreased the hippocampal pCREB/CREB ratio. The levels of BDNF in the hippocampus and the amygdala decreased under acute stress. Acute stress-induced increase of nicotine reward increased BDNF levels in the hippocampus. Moreover, the animals' exposure to the CPP apparatus without any drug administration increased the ratios of pCREB/tCREB and BDNF/ß-actin in the targeted sites. In summary, the present study indicate that the alterations of the ratio of pCREB/CREB and also the level of BDNF in the hippocampus may be critical for enhancing nicotine reward under stress condition. The evidence from this study suggests the distinct roles of the hippocampus and the amygdala in mediating nicotine reward under stress.

Mesenchymal Stromal Cell-Derived Small Extracellular Vesicles Induce Ischemic Neuroprotection by Modulating Leukocytes and Specifically Neutrophils.

Background and Purpose- Small extracellular vesicles (sEVs) obtained from mesenchymal stromal cells (MSCs) were shown to induce neurological recovery after focal cerebral ischemia in rodents and to reverse postischemic lymphopenia in peripheral blood. Since peripheral blood cells, especially polymorphonuclear neutrophils (PMNs), contribute to ischemic brain injury, we analyzed brain leukocyte responses to sEVs and investigated the role of PMNs in sEV-induced neuroprotection. Methods- Male C57Bl6/j mice were exposed to transient intraluminal middle cerebral artery occlusion. After reperfusion, vehicle or sEVs prepared from conditioned media of MSCs raised from bone marrow samples of 3 randomly selected healthy human donors were intravenously administered. sEVs obtained from normoxic and hypoxic MSCs were applied. PMNs were depleted in vehicle and MSC-sEV-treated mice. Neurological deficits, ischemic injury, blood-brain barrier integrity, peripheral blood leukocyte responses, and brain leukocyte infiltration were evaluated over 72 hours. Results- sEV preparations of all 3 donors collected from normoxic MSCs significantly reduced neurological deficits. Preparations of 2 of these donors significantly decreased infarct volume and neuronal injury. sEV-induced neuroprotection was consistently associated with a decreased brain infiltration of leukocytes, namely of PMNs, monocytes/macrophages, and lymphocytes. sEVs obtained from hypoxic MSCs (1% O2) had similar effects on neurological deficits and ischemic injury as MSC-sEVs obtained under regular conditions (21% O2) but also reduced serum IgG extravasation-a marker of blood-brain barrier permeability. PMN depletion mimicked the effects of MSC-sEVs on neurological recovery, ischemic injury, and brain PMN, monocyte, and lymphocyte counts. Combined MSC-sEV administration and PMN depletion did not have any effects superior to PMN depletion in any of the readouts examined. Conclusions- Leukocytes and specifically PMNs contribute to MSC-sEV-induced ischemic neuroprotection. Individual MSC-sEV preparations may differ in their neuroprotective activities. Potency assays are urgently needed to identify their therapeutic efficacy before clinical application. Visual Overview- An online visual overview is available for this article.

FoxP3 deficiency causes no inflammation or neurodegeneration in the murine brain.

Regulatory T cells (Treg) maintain immunological self-tolerance and their functional or numerical deficits are associated with progression of several neurological diseases. We examined the effects of Treg absence on the structure and integrity of the unchallenged murine brain. When compared to control, Treg-deficient FoxP3sf mutant mice showed no differences in brain size, myelin amount and oligodendrocyte numbers. FoxP3sf strain displayed no variations in quantity of neurons and astrocytes, whereas microglia numbers were slightly reduced. We demonstrate lack of neuroinflammation and parenchymal responses in the brains of Treg-deficient mice, suggesting a minor Treg role in absence of blood-brain barrier breakdown.

Dose-Dependent Microglial and Astrocytic Responses Associated With Post-ischemic Neuroprotection After Lipopolysaccharide-Induced Sepsis-Like State in Mice.

In contrast to lipopolysaccharide (LPS)-induced preconditioning, which has repeatedly been examined in the past, the effects of post-ischemic LPS-induced sepsis, although clinically considerably more important, have not systemically been studied. We exposed mice to transient intraluminal middle cerebral artery occlusion (MCAO) and examined the effects of intraperitoneal LPS (0.1 or 1 mg/kg) which was administered 24 h post-ischemia. Post-ischemic glial reactivity, neuronal survival and neurological outcome were differently modulated by the higher and the lower LPS dose. Although both doses promoted neuronal survival after 72 h, the underlying mechanisms were not similar. Mice receiving 1 mg/kg LPS exhibited transient hypothermia at 1 and 3 hours post sepsis (hps), followed by reduced focal neurological deficits at 24, 48 and 72 hps. The lower dose (0.1 mg/kg) did not induce hypothermia, but reduced microglia/macrophage activation with the appearance of an anti-inflammatory CD206 positive cell phenotype in the brain parenchyma. Together, our results indicate a novel, dose-dependent modulation of microglial cells that is intricately involved in brain protection.

Neuroprotection Induced by Energy and Protein-Energy Undernutrition Is Phase-Dependent After Focal Cerebral Ischemia in Mice.

Malnutrition predisposes to poor stroke outcome. In animal models, undernutrition protected against ischemic injury in some, but not in other studies. In view of diverse stroke models and food restriction paradigms, the consequences of undernutrition are poorly understood. Herein, we exposed mice to energy-reduced and protein-energy-reduced diets for 7-30 days and subsequently induced intraluminal middle cerebral artery occlusion. Undernutrition phase dependently influenced ischemic injury. Short-lasting 7 days of protein-energy undernutrition, but not energy undernutrition, decreased post-ischemic brain leukocyte infiltration and microglial activation and reduced brain Il-1ß mRNA, but did not protect against ischemic injury. Fourteen days of energy and protein-energy undernutrition, on the other hand, reduced ischemic injury despite absence of anti-inflammatory effects. Anti-oxidant genes (Sod-1, Sod-2, and Cat mRNAs) were regulated in the liver and, to a lesser extent, the ischemic brain, indicating an adapted, compensated stage. Conversely, 30 days of energy and protein-energy undernutrition caused progressive animal exhaustion associated with post-ischemic hypoperfusion, rise of metabolic markers (Sirt-1 and Glut-1 mRNAs, Sirt-1 protein) in the ischemic brain, and reregulation of pro- and anti-oxidant markers (now also Nox-4 and Gpx-3 mRNAs) in the liver. In the latter condition, no neuroprotection was noted. Our study suggests an adaptation of metabolic systems that provides neuroprotection in a circumscribed time window.

The involvement of ventral hippocampal microglial cells, but not cannabinoid CB1 receptors, in morphine-induced analgesia in rats.

It is well known that glial cells are involved in pain processing. The purpose of the present study was to investigate the possible involvement of the ventral hippocampal (VH) glial cells in morphine-induced analgesia. A tail-flick apparatus was used to measure pain sensitivity in male Wistar rats that were bilaterally cannulated in the VH by stereotaxic surgery. The results showed that intraperitoneal (i.p.) administration of morphine (2.5-7.5 mg/kg) induced analgesia in a time-dependent manner. The blockade of the VH glial cell activation by bilateral microinjection of a glial inhibitor, minocycline (5-15 µg/rat) into the VH with an ineffective dose of morphine (2.5 mg/kg, i.p) significantly increased morphine analgesia. Considering that the endocannabinoid system via CB1 receptors play a crucial role in pain modulation, we also assessed the possible role of the VH cannabinoid CB1 receptors in the functional interaction between minocycline and morphine in acute pain. Our results indicated that intra-VH injection of the cannabinoid CB1 receptor agonist, arachidonylcyclopropylamide (ACPA; 4-12 ng/rat) had no effect on minocycline-induced potentiation of morphine analgesia. It should be considered that intra-VH microinjection of minocycline or ACPA by itself had no effect on tail-flick latency. Our findings suggest that the activation of the VH microglial cells may be involved in mediating pain sensation, because the inhibition of these cells by intra-VH injection of minocycline could potentiate morphine-induced analgesia. Although endocannabinoids have a regulatory role in glia function, the activation of CB1 receptors could not affect the potentiative effect of minocycline on morphine analgesia.

Deactivation of ATP-Binding Cassette Transporters ABCB1 and ABCC1 Does Not Influence Post-ischemic Neurological Deficits, Secondary Neurodegeneration and Neurogenesis, but Induces Subtle Microglial Morphological Changes.

ATP-binding cassette (ABC) transporters prevent the access of pharmacological compounds to the ischemic brain, thereby impeding the efficacy of stroke therapies. ABC transporters can be deactivated by selective inhibitors, which potently increase the brain accumulation of drugs. Concerns have been raised that long-term ABC transporter deactivation may promote neuronal degeneration and, under conditions of ischemic stroke, compromise neurological recovery. To elucidate this issue, we exposed male C57BL/6 mice to transient intraluminal middle cerebral artery occlusion (MCAO) and examined the effects of the selective ABCB1 inhibitor tariquidar (8 mg/kg/day) or ABCC1 inhibitor MK-571 (10 mg/kg/day), which were administered alone or in combination with each other over up to 28 days, on neurological recovery and brain injury. Mice were sacrificed after 14, 28, or 56 days. The Clark score, RotaRod, tight rope, and open field tests revealed reproducible motor-coordination deficits in mice exposed to intraluminal MCAO, which were not influenced by ABCB1, ABCC1, or combined ABCB1 and ABCC1 deactivation. Brain volume, striatum volume, and corpus callosum thickness were not altered by ABCB1, ABCC1 or ABCB1, and ABCC1 inhibitors. Similarly, neuronal survival and reactive astrogliosis, evaluated by NeuN and GFAP immunohistochemistry in the ischemic striatum, were unchanged. Iba1 immunohistochemistry revealed no changes of the overall density of activated microglia in the ischemic striatum of ABC transporter inhibitor treated mice, but subtle changes of microglial morphology, that is, reduced microglial cell volume by ABCB1 deactivation after 14 and 28 days and reduced microglial ramification by ABCB1, ABCC1 and combined ABCB1 and ABCC1 deactivation after 56 days. Endogenous neurogenesis, assessed by BrdU incorporation analysis, was not influenced by ABCB1, ABCC1 or combined ABCB1 and ABCC1 deactivation. Taken together, this study could not detect any exacerbation of neurological deficits or brain injury after long-term ABC transporter deactivation in this preclinical stroke model.

Moderate Protein Restriction Protects Against Focal Cerebral Ischemia in Mice by Mechanisms Involving Anti-inflammatory and Anti-oxidant Responses.

Food composition influences stroke risk, but its effects on ischemic injury and neurological deficits are poorly examined. While severe reduction of protein content was found to aggravate neurological impairment and brain injury as a consequence of combined energy-protein malnutrition, moderate protein restriction not resulting in energy deprivation was recently suggested to protect against perinatal hypoxia-ischemia. Male C57BL6/j mice were exposed to moderate protein restriction by providing a normocaloric diet containing 8% protein (control: 20% protein) for 7, 14, or 30 days. Intraluminal middle cerebral artery occlusion was then induced. Mice were sacrificed 24 h later. Irrespective of the duration of food modification (that is, 7-30 days), protein restriction reduced neurological impairment of ischemic mice revealed by a global and focal deficit score. Prolonged protein restriction over 30 days also reduced infarct volume, brain edema, and blood-brain barrier permeability and increased the survival of NeuN+ neurons in the core of the stroke (i.e., striatum). Neuroprotection by prolonged protein restriction went along with reduced brain infiltration of CD45+ leukocytes and reduced expression of inducible NO synthase and interleukin-1ß. As potential mechanisms, increased levels of the NAD-dependent deacetylase sirtuin-1 and anti-oxidant glutathione peroxidase-3 were noted in ischemic brain tissue. Irrespective of the protein restriction duration, a shift from pro-oxidant oxidative stress markers (NADPH oxidase-4) to anti-oxidant markers (superoxide dismutase-1/2, glutathione peroxidase-3 and catalase) was found in the liver. Moderate protein restriction protects against ischemia in the adult brain. Accordingly, dietary modifications may be efficacious strategies promoting stroke outcome.

Postacute Delivery of GABAA α5 Antagonist Promotes Postischemic Neurological Recovery and Peri-infarct Brain Remodeling.

Background and Purpose- Poststroke, neuronal excitability is tonically reduced in peri-infarct tissue via inhibitory influences of extrasynaptic GABAA receptors. We hypothesized that GABAA α5 blockade by the competitive antagonist S44819 enhances postischemic neurological recovery, brain remodeling, and neuroplasticity. Methods- In an explorative study followed by a confirmation study, male C57Bl6/j mice were exposed to transient intraluminal middle cerebral artery occlusion. Starting 72 hours poststroke, vehicle or S44819 (3 or 10 mg/kg, BID) was delivered orally for 28 days. Neurological recovery, perilesional tissue remodeling, and contralesional pyramidal tract plasticity were evaluated for 42 days, that is, 14 days after completion of S44819 delivery. Results- S44819, delivered at 10 but not 3 mg/kg, persistently improved motor coordination and spatial memory in both studies. Striatal atrophy was reduced by 10 mg/kg S44819 at 42 days post-treatment onset, and neuronal long-term survival in the peri-infarct striatum was increased. Delayed neuroprotection was associated with reduced peri-infarct astrogliosis, increased peri-infarct brain capillary density, and increased neural precursor cell proliferation and differentiation in proximity to the ipsilesional subventricular zone. Contralesional pyramidal tract plasticity, evaluated by anterograde tract tracing at the level of the red nucleus, was not influenced by S44819. Concentrations of neurotrophic (brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor) and angiogenic (vascular endothelial growth factor and basic fibroblast growth factor) growth factors were elevated by 10 mg/kg S44819 in peri-infarct but not contralesional brain tissue. Conclusions- Our data demonstrate that S44819 enhances neurological recovery and peri-infarct brain remodeling in the postacute stroke phase.

Brain nicotinic acetylcholine receptors are involved in stress-induced potentiation of nicotine reward in rats.

The aim of the present study was to examine the possible role of nicotinic acetylcholine receptors of the dorsal hippocampus (CA1 regions), the medial prefrontal cortex or the basolateral amygdala in the effect of acute or sub-chronic stress on nicotine-induced conditioned place preference. Our results indicated that subcutaneous administration of nicotine (0.2 mg/kg) induced significant conditioned place preference. Exposure to acute or sub-chronic elevated platform stress potentiated the response of an ineffective dose of nicotine. Pre-conditioning intra-CA1 (0.5-4 µg/rat) or intra-medial prefrontal cortex (0.2-0.3 µg/rat) microinjection of mecamylamine (a non-selective nicotinic acetylcholine receptor antagonist) reversed acute stress-induced potentiation of nicotine reward as measured in the conditioned place preference paradigm. By contrast, pre-conditioning intra-basolateral amygdala microinjection of mecamylamine (4 µg/rat) potentiated the effects of acute stress on nicotine reward. Our findings also showed that intra-CA1 or intra-medial prefrontal cortex, but not intra-basolateral amygdala, microinjection of mecamylamine (4 µg/rat) prevented the effect of sub-chronic stress on nicotine reward. These findings suggest that exposure to elevated platform stress potentiates the rewarding effect of nicotine which may be associated with the involvement of nicotinic acetylcholine receptors. It seems that there is a different contribution of the basolateral amygdala, the medial prefrontal cortex or the CA1 nicotinic acetylcholine receptors in stress-induced potentiation of nicotine-induced conditioned place preference.