α7 Nicotinic Acetylcholine Receptor Stimulation Attenuates Neuroinflammation through JAK2-STAT3 Activation in Murine Models of Intracerebral Hemorrhage.

Accounting for high mortality and morbidity rates, intracerebral hemorrhage (ICH) remains one of the most detrimental stroke subtypes lacking a specific therapy. Neuroinflammation contributes to ICH-induced brain injury and is associated with unfavorable outcomes. This study aimed to evaluate whether α7 nicotinic acetylcholine receptor (α7nAChR) stimulation ameliorates neuroinflammation after ICH. Male CD-1 mice and Sprague-Dawley were subjected to intracerebral injection of autologous blood or bacterial collagenase. ICH animals received either α7nAChR agonist PHA-543613 alone or combined with α7nAChR antagonist methyllycaconitine (MLA) or Janus kinase 2 (JAK2) antagonist AG490. Neurobehavioral deficits were evaluated at 24 hours, 72 hours, and 10 weeks after ICH induction. Perihematomal expressions of JAK2, signal transducer and activator of transcription 3 (STAT3), tumor necrosis factor-α (TNF-α), and myeloperoxidase (MPO) were quantified via Western blot. Histologic volumetric analysis of brain tissues was conducted after 10 weeks following ICH induction. PHA-543613 improved short-term neurobehavioral (sensorimotor) deficits and increased activated perihematomal JAK2 and STAT3 expressions while decreasing TNF-α and MPO expressions after ICH. MLA reversed these treatment effects. PHA-543613 also improved long-term neurobehavioral (sensorimotor, learning, and memory) deficits and ameliorated brain atrophy after ICH. These treatment effects were reduced by AG490. α7nAChR stimulation reduced neuroinflammation via activation of the JAK2-STAT3 pathway, thereby ameliorating the short- and long-term sequelae after ICH.

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.

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.

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.

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.

Dual effects of melatonin on oxidative stress after surgical brain injury in rats.

The purpose of this study was to evaluate the effect of melatonin on oxidative stress occurring in the brain after routine lobectomy neurosurgery procedures. Different concentrations of melatonin (5, 15 and 150 mg/kg) were administered 1 hr before lobectomy in a rodent surgical brain injury (SBI) model. Neurological outcomes were assessed 24 hr before the killing of the rodents, for evaluation of brain water content (brain edema) and lipid peroxidation (oxidative stress). The results showed that lower doses (5 and 15 mg/kg) failed to reduce brain edema, but the 15 mg/kg dose did lower oxidative stress and improved several neurological parameters. High concentration of melatonin (150 mg/kg) significantly increased brain edema and elevated oxidative stress when compared with the vehicle-treated group. Furthermore, high-dose melatonin also worsened neurological outcomes compared with other groups. The study suggests that melatonin has dual effects: low-dose melatonin may provide neuroprotective effects against SBI but a high dose may aggravate some parameters after SBI.

The antioxidant effects of melatonin in surgical brain injury in rats.

BACKGROUND: Surgical brain injury (SBI) to normal brain tissue can occur as inevitable sequelae of neurosurgical operations. SBI can contribute to post-operative complications such as brain edema following blood-brain barrier (BBB) disruption leading to neurological deficits. Melatonin is a commonly used drug with known antioxidant properties and neuroprotective effects in experimental animal studies (Chen et al., J Pineal Res 41:175-182, 2006; Chen et al., J Pineal Res 40(3):242-250, 2006; Cheung, J Pineal Res 34:153-160, 2003; Lee et al., J Pineal Res 42(3):297-309, 2007; Reiter et al., Exp Biol Med (Maywood) 230(2):104-117, 2005). METHODS: We tested different concentrations of melatonin (5 mg/kg, 15 mg/kg and 150 mg/kg) administered 1 hour before surgery for neuroprotection against SBI using a rodent model. Post-operative assessment included brain water content (brain edema), lipid peroxidation assays (oxidative stress), and neurological assessment. FINDINGS: The results showed a trend in decreasing brain edema with lower doses of melatonin (5 mg/kg and 15 mg/ kg), however, high concentration of melatonin (150 mg/kg) significantly increased brain edema compared to all other groups. This deleterious effect of high-dose melatonin was also observed in lipid-peroxidation assay wherein lower-dose melatonin (15 mg/kg) attenuated oxidative stress, but high-dose melatonin (150 mg/kg) increased oxidative stress as compared to vehicle-treated group. Furthermore, high-dose melatonin also worsened neurological outcomes compared to other groups whereas; the low-dose melatonin group (15 mg/kg) showed some improved neurological parameters. CONCLUSIONS: The study suggests that low-dose melatonin may provide neuroprotective effects against SBI. Further studies are needed to confirm this. More importantly, the findings of the study stress the need to carefully reassess safety issues with high doses of melatonin, which is considered to be a practically non-toxic drug.