Occlusion of the MCA by an intraluminal filament may cause disturbances in the hippocampal blood flow due to anomalies of circle of Willis and filament thickness

We examined blood flow changes and histology in the hippocampus induced by occlusion of the middle cerebral artery (MCA) by a filament in Swiss albino and SV-129 mice (n=67) and in Wistar rats (n=64). Filling cerebral arteries with carbon black revealed that one or both posterior communicating arteries were hypoplastic in 50% of Swiss mice. Ischemic changes were detected in the ipsilateral hippocampus with 2,3,5-triphenyl tetrazolium chloride or hematoxylin and eosin staining when these mice were subjected to 2-h MCA occlusion and 22-h reperfusion. No such abnormalities were found in SV-129 mice and Wistar rats (except one). The hippocampal blood flow dropped to 60+/-2.3% of the baseline in mice with a normal circle of Willis but to 37+/-4.2% in those with an incomplete circle when the MCA was occluded with a 6/0 nylon filament. When an 8/0 filament was used, no flow change in mice with a normal circle but a decrease to 60+/-2% in those with an incomplete circle was observed. A flow drop to 63+/-4% was also seen in Wistar rats when a 3/0 filament used. These data demonstrate that occlusion of the MCA by a thick filament may cause flow reduction in the hippocampus, which may be severe enough to lead to infarction if the circle of Willis is anomalous.     

Transcorneal stimulation of trigeminal nerve afferents to increase cerebral blood flow in rats with cerebral vasospasm: a noninvasive method to activate the trigeminovascular reflex

OBJECT: The goal of this study was to investigate whether stimulation of trigeminal afferents in the cornea could enhance cerebral blood flow (CBF) in rats after they have been subjected to experimental subarachnoid hemorrhage (SAH). Cerebral vasospasm following SAH may compromise CBF and increase the risks of morbidity and mortality. Currently, there is no effective treatment for SAH-induced vasospasm. Direct stimulation of the trigeminal nerve has been shown to dilate constricted cerebral arteries after SAH; however, a noninvasive method to activate this nerve would be preferable for human applications. The authors hypothesized that stimulation of free nerve endings of trigeminal sensory fibers in the face might be as effective as direct stimulation of the trigeminal nerve. METHODS: Autologous blood obtained from the tail artery was injected into the cisterna magna of 10 rats. Forty-eight and 96 hours later (five rats each) trigeminal afferents were stimulated selectively by applying transcorneal biphasic pulses (1 msec, 3 mA, and 30 Hz), and CBF enhancements were detected using laser Doppler flowmetry in the territory of the middle cerebral artery. Stimulation-induced changes in cerebrovascular parameters were compared with similar parameters in sham-operated controls (six rats). Development of vasospasm was histologically verified in every rat with SAH. Corneal stimulation caused an increase in CBF and blood pressure and a net decrease in cerebrovascular resistance. There were no significant differences between groups for these changes. CONCLUSIONS: Data from the present study demonstrate that transcorneal stimulation of trigeminal nerve endings induces vasodilation and a robust increase in CBF. The vasodilatory response of cerebral vessels to trigeminal activation is retained after SAH-induced vasospasm.     

Histone Deacetylase Inhibition Activity and Molecular Docking of (E )‐Resveratrol: Its Therapeutic Potential in Spinal Muscular Atrophy

Spinal muscular atrophy is an autosomal recessive motor neuron disease that is caused by mutation of the survival motor neuron gene (SMN1) but all patients retain a nearly identical copy, SMN2. The disease severity correlates inversely with increased SMN2 copy. Currently, the most promising therapeutic strategy for spinal muscular atrophy is induction of SMN2 gene expression by histone deacetylase inhibitors. Polyphenols are known for protection against oxidative stress and degenerative diseases. Among our candidate prodrug library, we found that (E )‐resveratrol, which is one of the polyphenolic compounds, inhibited histone deacetylase activity in a concentration‐dependent manner and half‐maximum inhibition was observed at 650 μm . Molecular docking studies showed that (E )‐resveratrol had more favorable free energy of binding (?9.09 kcal/mol) and inhibition constant values (0.219 μm ) than known inhibitors. To evaluate the effect of (E )‐resveratrol on SMN2 expression, spinal muscular atrophy type I fibroblast cell lines was treated with (E )‐resveratrol. The level of full‐length SMN2 mRNA and protein showed 1.2‐ to 1.3‐fold increase after treatment with 100 μm (E )‐resveratrol in only one cell line. These results indicate that response to (E )‐resveratrol treatment is variable among cell lines. This data demonstrate a novel activity of (E )‐resveratrol and that it could be a promising candidate for the treatment of spinal muscular atrophy.     

Loss of NeuN immunoreactivity after cerebral ischemia does not indicate neuronal cell loss: a cautionary note

NeuN immunoreactivity is used as a specific marker for neurons. The number of NeuN-positive cells decreases under pathological conditions. This finding is usually considered as an evidence of neuronal loss. However, decrease in NeuN labeling may also be caused by depletion of the protein or loss of its antigenicity. Hence, we have investigated the morphological features of neurons that lost NeuN immunoreactivity and the NeuN protein levels in mouse brain after cerebral ischemia. The number of NeuN-labeled cells was decreased 6 h after a mild ischemic insult (30 min middle cerebral artery occlusion) in penumbral and core regions. Hematoxylin and eosin (H&E) staining of adjacent sections showed that neurons in the penumbra were not disintegrated but displayed early ischemic changes. The nuclear NeuN staining was dramatically reduced or lost in some neurons. However, Hoechst 33258 staining of the same sections revealed that these nuclei were preserved with an intact membrane. Labeling of neurons that had lost NeuN-positivity with antibodies against caspase-3-p20, which is constitutively not present but emerges in neurons after ischemia, disclosed that these neurons still preserved their integrity. Moreover, Western blots showed that NeuN protein levels were not decreased, suggesting that reduced NeuN antigenicity accounted for loss of immunoreactivity in this mild brain injury model. Supporting this idea, NeuN labeling was partially restored after antigenic retrieval. In conclusion, since NeuN immunoreactivity readily decreases after metabolic perturbations, reduced NeuN labeling should not be taken as an indicator of neuronal loss and, quantitative analysis based on NeuN-positivity should be used cautiously after central nervous system (CNS) injury.     

The effects of myasthenic immunoglobulins on neuromuscular transmission and thymus histology in mice

An autoimmune reaction to the endplate acetylcholine receptor (AChR) is thought to be responsible for the muscular weakness of myasthenia gravis (MG). However, the significance of antithymic antibodies and the thymic AChR-like protein is still uncertain. We transferred immunoglobulins (Igs) from patients with MG to mice. Thymitis was observed in 12 of 14 mice, and miniature endplate potential amplitudes were reduced in 8. Control mice showed none of these abnormalities. Immunofluorescence examination failed to reveal the binding of human IgG, IgM, or IgA to the thymic tissue. Our findings support the hypothesis that antithymic antibodies may alter thymic histology.     

Endothelial nitric oxide synthase-dependent cerebral blood flow augmentation by L-arginine after chronic statin treatment.

Nitric oxide, a product of nitric oxide synthase activity, relaxes vascular smooth muscle and elevates brain blood flow. We evaluated the importance of eNOS to cerebral blood flow augmentation after L-arginine infusion and increases in flow after eNOS upregulation in SV-129 mice. Blood flow was measured by laser-Doppler flowmetry before and after L-arginine infusion (450 mg/kg during a 15-minute period) or measured by 14C-iodoamphetamine indicator fractionation or 14C-iodoantipyrine tissue equilibration techniques. rCBF increased by 26% (laser Doppler flowmetry) after L-arginine infusion but did not change in mutant mice deficient in eNOS expression. After eNOS upregulation by chronic simvastatin treatment (2 mg/kg subcutaneously, daily for 14 days), L-arginine amplified and sustained the hyperemia (38%) and increased absolute brain blood flow from 86 +/- 7 to 119 +/- 10 mL/100 g per minute. Furthermore, pretreatment with simvastatin enhanced blood flow within ischemic brain tissue after middle cerebral artery occlusion. Together, these findings suggest that eNOS activity is critical for blood flow augmentation during acute L-arginine infusion, and chronic eNOS upregulation combined with L-arginine administration provides a novel strategy to elevate cerebral blood flow in the normal and ischemic brain.     

Nitric oxide is involved in ischemia-induced apoptosis in brain: a study in neuronal nitric oxide synthase null mice

Nitric oxide can promote or inhibit apoptosis depending on the cell type and coexisting metabolic or experimental conditions. We examined the impact of nitric oxide on development of apoptosis 6, 24, and 72 h after permanent middle cerebral artery occlusion in mutant mice that lack the ability to generate nitric oxide from neuronal nitric oxide synthase. Adjacent coronal sections passing through the anterior commissure were stained with hematoxylin and eosin or terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). Immunoblotting was used to identify changes in the anti- and proapoptotic proteins Bcl-2 and Bax, respectively. Activation of caspases was assessed by appearance of actin cleavage products using a novel antiserum directed against 32-kDa actin fragment (fractin). In the neuronal nitric oxide synthase mutant mouse, infarct size and TUNEL positive apoptotic neurons were reduced compared to the wild-type controls. At 6 h, Bcl-2 levels in the ischemic hemisphere were increased in mutants but decreased in the wild-type strain. Bax levels did not change significantly. Caspase-mediated actin cleavage appeared in the ischemic hemisphere at this time point, and was significantly less in mutant brains at 72 h compared to the wild-type. The reduction in the number of TUNEL and fractin positive apoptotic cells appears far greater than anticipated based on the smaller lesion size in mutant mice.Hence, from these data we suggest that a deficiency in neuronal nitric oxide production slows the development of apoptotic cell death after ischemic injury and is associated with preserved Bcl-2 levels and delayed activation of effector caspases.