Neuroprotective effects of methylene blue in streptozotocin-induced model of Alzheimer's disease.

Methylene blue (MB) can be used as a multidirectional neuroprotector to stop the development of multiple cascades of neuron damage during neurodegenerative processes. This study assesses a protective effect of MB, using an experimental simulation of sporadic Alzheimer's disease by intracerebroventricular administration of streptozotocin (STZ) in rats. It was found that a STZ-induced impairment of memory can be partially mitigated with intravenous injections of MB after the administration of STZ. The treatment of animals with MB prevented the STZ-induced increase in the number and density of microglial and GFAP-positive cells in the brain cortex. In addition, it was shown that the expression of the LC3B protein, an indicator of autophagy, increases in the hippocampus of animals treated with STZ. In the hippocampus of animals treated with MB, an increase in the expression of the LC3B protein was prevented. Using the Griess reaction assay and immunocytochemical study was found that MB reduces lipopolysaccharide-induced NO-production and the expression of iNOS in cultured neurons. In conclusion, our data demonstrate that MB has neuroprotective and anti-inflammatory effects and is able to prevent autophagy. These effects have important therapeutic implications, so MB could potentially play a role in the treatment of neurodegenerative processes.

The Delayed Neuroprotective Effect of Methylene Blue in Experimental Rat Brain Trauma.

After traumatic brain injury (TBI), an increase in dysfunction of the limbs contralateral to injury focus was observed. Using different behavioral tests, we found that a single intravenous injection of methylene blue (MB, 1 mg/kg) 30 min after the injury reduced the impairment of the motor functions of the limbs from 7 to 120 days after TBI. Administration of methylene blue 30 min after the injury and then monthly (six injections in total) was the most effective both in terms of preservation of limb function and duration of therapeutic action. This therapeutic effect was clearly manifested from the seventh day and continued until the end of the experiment-by the 180th day after TBI. MB is known to possess antioxidant properties; it has a protective effect against TBI by promoting autophagy and minimizing lesion volume in the first two weeks after TBI. Studies of the brains on the 180th day after TBI demonstrated that the monthly treatment of animals with MB statistically significantly prevented an increase in the density of microglial cells in the ipsilateral hemisphere and a decrease in the thickness of the corpus callosum in the contralateral hemisphere in comparison with untreated animals. However, on the 180th day after TBI, the magnetic resonance imaging scan of the animal brains did not show a significant reduction in the volume of the lesion in MB-treated animals. These findings are important for understanding the development of the long-term effects of TBI and expand the required therapeutic window for targeted neuroprotective interventions.

The single intravenous administration of methylene blue after traumatic brain injury diminishes neurological deficit, blood-brain barrier disruption and decrease in the expression of S100 protein in rats.

The protective effect of methylene blue (MB) was investigated on the model of focal one-sided traumatic brain injury (TBI) of the sensorimotor cortex region from 1 to 7 days after the injury. TBI caused a reliable disruption of the functions of the limbs contralateral to injury focus, an increase in the expression of S100 protein and blood-brain barrier (BBB) permeability in the ipsilateral hemisphere. The single intravenous injection of MB (1 mg/kg body weight) 30 min after TBI significantly reduced the limb function impairment as well as a TBI-induced increase in the expression of inflammatory marker S100 protein, and BBB permeability. When modeling inflammation in vitro, MB was found to protect cultured neurons from the toxic effects of lipopolysaccharide. In conclusion, the preservation of blood-brain barrier and a decrease in the expression of S100 protein may be an important mechanism by means of which MB improves neurological outcome. Our data demonstrate that MB can be a very promising pharmacological compound with neuroprotective properties for TBI treatment.

The single intravenous administration of mitochondria-targeted antioxidant SkQR1 after traumatic brain injury attenuates neurological deficit in rats.

The protective effect of SkQR1, a mitochondria-targeted antioxidant, was investigated on the model of focal one-sided traumatic brain injury (TBI) of the sensorimotor cortex region from 1 to 7 days after the injury. TBI caused a reliable disruption of the functions of the limbs contralateral to injury focus. The intravenous single injection of SkQR1 (250 nmol/kg) but not C12R1 (a SkQR1 homologue devoid of the antioxidant group) 30 min after TBI reduced the impairment of the motor functions of the limbs. A statistically significant improvement in limb function in animals was shown using 3 different tests: limb-placing test, beam-walking test and grip strength test. A pronounced therapeutic effect appeared on the 1th day and lasted until the end of the experiment - the 7th day after TBI. Histopathological examination showed that in the group of animals that did not receive SkQR1 in the marginal layer of the lesion there was a marked increase in astroglial expression, infiltration with segmented neutrophils, and poor survivability of neurons compared with animals treated with SkQR1. The obtained results demonstrate that the single use of plastoquinone-containing mitochondria-targeted antioxidant SkQR1 at the early stages of development of traumatic brain damage can reduce TBI-related disruptions of limb functions, and that mechanisms of the brain damage after trauma are dependent on the production of mitochondrial reactive oxygen species.

The delayed protective effect of GK-2, а dipeptide mimetic of Nerve Growth Factor, in a model of rat traumatic brain injury.

The delayed protective effect of GK-2, a dipeptide mimetic of Nerve Growth Factor, was investigated on the model of focal one-sided traumatic brain injury (TBI) of the sensorimotor cortex region on the 180th day after the injury. TBI caused a reliably disruption of the functions of the limbs contralateral to injury focus. The intraperitoneal administration of GK-2 (1 mg/kg) from 1st to 4th and from 7th to 10th days after TBI reduced the impairment of the motor functions of the limbs. This therapeutic effect significant manifested itself from the 7th day and continued until the end of the experiment - on the 180th day after TBI. Morphological studies of the animal brains on the 180th day after TBI demonstrated a decrease in the number of neurons in the V layer of the cerebral cortex and a decrease in the thickness of the corpus callosum. The treatment of animals with GK-2 after TBI statistically significant prevented a decrease in the density of neurons in the ipsilateral hemisphere and a decrease in the thickness of the corpus callosum in the contralateral hemisphere in comparison with untreated animals. Additionally, we showed in vitro that GK-2 exhibits neuroprotective properties under oxidative stress in primary hippocampal cultures. Our results demonstrate that the use of GK-2 at the early stages of development of traumatic brain damage can prevent such delayed damage as neuronal and axonal degeneration as well as reduce TBI-related disruptions of brain functions.

Streptozotocin toxicity in vitro depends on maturity of neurons.

Streptozotocin (STZ) is a glucosamine-nitrosourea compound that is particularly toxic to the insulin-producing beta-cells of the pancreas in mammals; it is used for experimental simulation of sporadic Alzheimer's disease by means of intracerebroventricular administration in vivo. Here we show that the application of 3-4 mM STZ to primary culture for 48 h induces neuronal death in immature (2-3 days in vitro) cultures of rat cerebellar granule cells. Mature cultures (7-8 days in vitro) were poorly sensitive to this toxic treatment. Immature cultures demonstrated a high expression of the protein PSA-NCAM, the marker of immature neurons, and they were insensitive to the toxic effect of glutamate. In mature cultures, this protein was poorly expressed, whereas neurons showed a very high sensitivity to the toxic effect of glutamate. Measurements of the concentration of intracellular free calcium ions ([Ca2+]i) showed that the STZ-induced [Ca2+]i increase in young neurons was six times higher than that in mature neurons. Our results show that STZ is very toxic for immature neurons and probably it can significantly impair neurogenesis.