Functional regulation of microglia by vitamin B12 alleviates ischemic stroke-induced neuroinflammation in mice.

Ischemic stroke is the second leading cause of death and disability worldwide, and efforts to prevent stroke, mitigate secondary neurological damage, and promote neurological recovery remain paramount. Recent findings highlight the critical importance of microbiome-related metabolites, including vitamin B12 (VB12), in alleviating toxic stroke-associated neuroinflammation. Here, we showed that VB12 tonically programmed genes supporting microglial cell division and activation and critically controlled cellular fatty acid metabolism in homeostasis. Intriguingly, VB12 promoted mitochondrial transcriptional and metabolic activities and significantly restricted stroke-associated gene alterations in microglia. Furthermore, VB12 differentially altered the functions of microglial subsets during the acute phase of ischemic stroke, resulting in reduced brain damage and improved neurological function. Pharmacological depletion of microglia before ischemic stroke abolished VB12-mediated neurological improvement. Thus, our preclinical studies highlight the relevance of VB12 in the functional programming of microglia to alleviate neuroinflammation, minimize ischemic injury, and improve host neurological recovery after ischemic stroke.

Neuroprotective Roles of the Adenosine A3 Receptor Agonist AST-004 in Mouse Model of Traumatic Brain Injury.

Traumatic brain injury (TBI) remains one of the greatest public health concerns with increasing morbidity and mortality rates worldwide. Our group reported that stimulation of astrocyte mitochondrial metabolism by P2Y1 receptor agonists significantly reduced cerebral edema and reactive gliosis in a TBI model. Subsequent data on the pharmacokinetics (PK) and rapid metabolism of these compounds suggested that neuroprotection was likely mediated by a metabolite, AST-004, which binding data indicated was an adenosine A3 receptor (A3R) agonist. The neuroprotective efficacy of AST-004 was tested in a control closed cortical injury (CCCI) model of TBI in mice. Twenty-four (24) hours post-injury, mice subjected to CCCI and treated with AST-004 (0.22 mg/kg, injected 30 min post-trauma) exhibited significantly less secondary brain injury. These effects were quantified with less cell death (PSVue794 fluorescence) and loss of blood brain barrier breakdown (Evans blue extravasation assay), compared to vehicle-treated TBI mice. TBI-treated mice also exhibited significantly reduced neuroinflammatory markers, glial-fibrillary acidic protein (GFAP, astrogliosis) and ionized Ca2+-binding adaptor molecule 1 (Iba1, microgliosis), both at the mRNA (qRT-PCR) and protein (Western blot and immunofluorescence) levels, respectively. Four (4) weeks post-injury, both male and female TBI mice presented a significant reduction in freezing behavior during contextual fear conditioning (after foot shock). AST-004 treatment prevented this TBI-induced impairment in male mice, but did not significantly affect impairment in female mice. Impairment of spatial memory, assessed 24 and 48 h after the initial fear conditioning, was also reduced in AST-004-treated TBI-male mice. Female TBI mice did not exhibit memory impairment 24 and 48 h after contextual fear conditioning and similarly, AST-004-treated female TBI mice were comparable to sham mice. Finally, AST-004 treatments were found to increase in vivo ATP production in astrocytes (GFAP-targeted luciferase activity), consistent with the proposed mechanism of action. These data reveal AST-004 as a novel A3R agonist that increases astrocyte energy production and enhances their neuroprotective efficacy after brain injury.

Prevention of brain damage after traumatic brain injury by pharmacological enhancement of KCNQ (Kv7, "M-type") K+ currents in neurons.

Nearly three million people in the USA suffer traumatic brain injury (TBI) yearly; however, there are no pre- or post-TBI treatment options available. KCNQ2-5 voltage-gated K+ channels underlie the neuronal "M current", which plays a dominant role in the regulation of neuronal excitability. Our strategy towards prevention of TBI-induced brain damage is predicated on the suggested hyper-excitability of neurons induced by TBIs, and the decrease in neuronal excitation upon pharmacological augmentation of M/KCNQ K+ currents. Seizures are very common after a TBI, making further seizures and development of epilepsy disease more likely. Our hypothesis is that TBI-induced hyperexcitability and ischemia/hypoxia lead to metabolic stress, cell death and a maladaptive inflammatory response that causes further downstream morbidity. Using the mouse controlled closed-cortical impact blunt TBI model, we found that systemic administration of the prototype M-channel "opener", retigabine (RTG), 30 min after TBI, reduces the post-TBI cascade of events, including spontaneous seizures, enhanced susceptibility to chemo-convulsants, metabolic stress, inflammatory responses, blood-brain barrier breakdown, and cell death. This work suggests that acutely reducing neuronal excitability and energy demand via M-current enhancement may be a novel model of therapeutic intervention against post-TBI brain damage and dysfunction.

Role of tumor necrosis factor-alpha and matrix metalloproteinase-9 in blood-brain barrier disruption after peripheral thermal injury in rats.

OBJECT: A relationship has been found between peripheral thermal injury and cerebral complications leading to injury and death. In the present study, the authors examined whether tumor necrosis factor-alpha (TNF-alpha) and matrix metalloproteinase-9 (MMP-9) play a causative role in blood-brain barrier (BBB) disruption after peripheral thermal injury. METHODS: Thirty-two male Sprague-Dawley rats were subjected to thermal injury. One hour later, 8 rats were injected with TNF-alpha neutralizing antibody, and 8 were injected with doxycycline, an inhibitor of the MMP family proteins; 16 rats did not receive any treatment. Brain tissue samples obtained 7 hours after injury in the treated animals were examined for BBB function by using fluorescein isothiocyanate-dextran and by assessing parenchymal water content. Protein expression of basement membrane components (collagen IV, laminin, and fibronectin) was quantified on Western blot analysis, and MMP-9 protein expression and enzyme activity were determined using Western blot and gelatin zymography. Thermally injured rats that did not receive treatment were killed at 3, 7, or 24 hours after injury and tested for BBB functioning at each time point. Histological analysis for basement membrane proteins was also conducted in untreated rats killed at 7 hours after injury. Results of testing in injured rats were compared with those obtained in a control group of rats that did not undergo thermal injury. RESULTS: At 7 hours after thermal injury, a significant increase in the fluorescein isothiocyanate-dextran and water content of the brain was found (p < 0.05), but BBB dysfunction was significantly decreased in the rats that received TNF-alpha antibody or doxycycline (p < 0.05). In addition, the components of the basal lamina were significantly decreased at 7 hours after thermal injury (p < 0.01), and there were significant increases in MMP-9 protein expression and enzyme activity (p < 0.05). The basal lamina damage was reversed by inhibition of TNF-alpha and MMP-9, and the increase in MMP-9 protein was reduced in the presence of doxycycline (p < 0.05). The authors found that MMP-9 enzyme activity was significantly increased after thermal injury (p < 0.01) but decreased in the presence of either TNF-alpha antibody or doxycycline (p < 0.01). CONCLUSIONS: The dual, inhibitory activity of both TNF-alpha and MMP-9 in brain injury suggests that a TNF-alpha and MMP-9 cascade may play a key role in BBB disruption. These results offer a better understanding of the pathophysiology of burn injuries, which may open new avenues for burn treatment beyond the level of current therapies.

Intranasal delivery of erythropoietin plus insulin-like growth factor-I for acute neuroprotection in stroke. Laboratory investigation.

OBJECT: Individually, the cytokines erythropoietin (EPO) and insulin-like growth factor-I (IGF-I) have both been shown to reduce neuronal damage significantly in rodent models of cerebral ischemia. The authors have previously shown that EPO and IGF-I, when administered together, provide acute and prolonged neuroprotection in cerebrocortical cultures against N-methyl-D-aspartate-induced apoptosis. The aim of this study was to determine whether intranasally applied EPO plus IGF-I can provide acute neuroprotection in an animal stroke model and to show that intranasal administration is more efficient at delivering EPO plus IGF-I to the brain when compared with intravenous, subcutaneous, or intraperitoneal administration. METHODS: The EPO and IGF-I were administered intranasally to mice that underwent transient middle cerebral artery occlusion (MCAO). Stroke volumes were measured after 1 hour of MCAO and 24 hours of reperfusion. To evaluate the long-term effects of this treatment, behavioral outcomes were assessed at 3, 30, 60, and 90 days following MCAO. Radiography and liquid scintillation were used to visualize and quantify the uptake of radiolabeled 125I-EPO and 125I-IGF-I into the mouse brain after intranasal, intravenous, subcutaneous, or intraperitoneal administration. RESULTS: Intranasal administration of EPO plus IGF-I reduced stroke volumes within 24 hours and improved neurological function in mice up to 90 days after MCAO. The 125I-EPO and 125I-IGF-I were found in the brain within 20 minutes after intranasal administration and accumulated within the injured areas of the brain. In addition, intranasal administration delivered significantly higher levels of the applied 125I-EPO and 125I-IGF-I to the brain compared with intravenous, subcutaneous, or intraperitoneal administration. CONCLUSIONS: The data demonstrate that intranasal EPO plus IGF-I penetrates into the brain more efficiently than other drug delivery methods and could potentially provide a fast and efficient treatment to prevent chronic effects of stroke.

Peripheral thermal injury causes blood-brain barrier dysfunction and matrix metalloproteinase (MMP) expression in rat.

Mortality after serious systemic thermal injury may be linked to significant increases in cerebral vascular permeability and edema due to blood-brain barrier (BBB) breakdown. This BBB disruption is thought to be mediated by a family of proteolytic enzymes known as matrix metalloproteinases (MMPs). The gelatinases, MMP-2 and MMP-9, digest the endothelial basal lamina of the BBB, which is essential for maintaining BBB integrity. The current study investigated whether disruption of microvascular integrity in a rat thermal injury model is associated with gelatinase expression and activity. Seventy-two adult Sprague-Dawley rats were anesthetized and submerged horizontally, in the supine position, in 100 degrees C (37 degrees C for controls) water for 6 s producing a third-degree burn affecting 60-70% of the total body surface area. Brain edema was detected by calculating water content. Real time PCR, Western blot, and zymography were used to quantify MMP mRNA, protein, and enzyme activity levels. Each group was quantified at 3, 7, 24, and 72 h post thermal injury. Brain water content was significantly increased 7 through 72 h after burn. Expression of brain MMP-9 mRNA was significantly increased as early as 3 h after thermal injury compared to controls, remained at 7 h (p<0.01), and returned to control levels by 24 h. MMP-9 protein levels and enzyme activity began to increase at 7 h and reached significant levels between 7 and 24 h after thermal injury. While MMP-9 protein levels continued to increase significantly through 72 h, enzyme activity returned to control level. The increase in MMP-9 expression and activity, associated with increased BBB permeability following thermal injury, indicates that MMP-9 may contribute to observed cerebral edema in peripheral thermal injury.

Peripheral thermal injury causes early blood-brain barrier dysfunction and matrix metalloproteinase expression in rat.

High mortality incidence after serious systemic thermal injury is believed to be linked to significant increases in cerebral permeability, ultimately leading to irreversible blood-brain barrier (BBB) breakdown. The aim of this study was to investigate whether disruption of microvascular integrity in a rat thermal injury model is associated with early matrix metalloproteinase (MMP) expression. A total of 35 Sprague-Dawley rats were studied in thermal injury and control groups, each group containing two subgroups, one for brain edema and Evans blue analysis and another for MMP mRNA analysis. Thermally injured animals were anesthetized and submerged vertically in 85 degrees C water to the neck for 6 seconds producing a third degree burn affecting 70% of the total body surface area. BBB integrity was determined by measuring amount of Evans blue after 7 hours of injury with a spectrophotometer. Brain edema was detected by calculating water content. Brain mRNA levels were determined with real-time PCR 3 and 7 hours post-injury. Brain water content was significantly increased after peripheral injury at hour 7. Evans blue leakage was also significantly increased at the same time, suggesting an impaired BBB function after injury. Expressions of MMP-2 and MMP-9 mRNA in brain were increased as early as 3 hours after injury and remained at hour 7. Our study demonstrated a significant increase in cerebral permeability that occurs after serious systemic thermal injury. The underlying mechanisms could be related to early expression of MMPs.

Reversal of attenuation of cerebrovascular reactivity to hypercapnia by a nitric oxide donor after controlled cortical impact in a rat model of traumatic brain injury.

OBJECT: Traumatic brain injury (TBI) attenuates the cerebral vasodilation to hypercapnia. Cortical spreading depression (CSD) also transiently reduces hypercapnic vasodilation. The authors sought to determine whether the CSD elicited by a controlled cortical impact (CCI) injury masks the true effect of TBI on hypercapnic vasodilation, and whether a nitric oxide (NO) donor can reverse the attenuation of hypercapnic vasodilation following CCI. METHODS: Anesthetized rats underwent moderate CCI. Cerebral blood flow was monitored with laser Doppler flowmetry and the response to hypercapnia was determined for injured and sham-injured animals. The effect of the NO donor, S-nitroso-N-acetylpenicillamine (SNAP), on this response was also assessed. At an uninjured cortical site ipsilateral to the CCI, a single wave of CSD was recorded and the CO2 response at this location was significantly attenuated for up to 30 minutes (seven rats, p < 0.05). At the injured cortex, hypercapnic vasodilation continued to be attenuated for 7 hours. The cerebral vasodilation to CO2 was 37 +/- 5% in injured rats (six) compared with 84 +/- 10% in the sham-injured group (five rats, p < 0.05). After 30 minutes of topical superfusion with SNAP, hypercapnic vasodilation was restored to 74 +/- 7% (nine rats, p > 0.1 compared with that in the sham-injured group). In contrast, papaverine, an NO-independent vasodilator, failed to reverse the attenuation of the CO2 response to CCI. CONCLUSIONS: The authors conclude that CSD elicited by CCI can mask the true effect of TBI on hypercapnic vasodilation for at least 30 minutes. Exogenous NO, but not papaverine, can reverse the attenuation of cerebrovascular reactivity to CO2 caused by TBI. This result supports the hypothesis that NO production is reduced after TBI and that the NO donor has a potential beneficial role in the clinical management of head injury.