ro-16-0154 and Disease-Models--Animal

When Zika virus emerged as a public health emergency there were no drugs or vaccines approved for its prevention or treatment. We used a high-throughput screen for Zika virus protease inhibitors to identify several inhibitors of Zika virus infection. We expressed the NS2B-NS3 Zika virus protease and conducted a biochemical screen for small-molecule inhibitors. A quantitative structure-activity relationship model was employed to virtually screen ∼138,000 compounds, which increased the identification of active compounds, while decreasing screening time and resources. Candidate inhibitors were validated in several viral infection assays. Small molecules with favorable clinical profiles, especially the five-lipoxygenase-activating protein inhibitor, MK-591, inhibited the Zika virus protease and infection in neural stem cells. Members of the tetracycline family of antibiotics were more potent inhibitors of Zika virus infection than the protease, suggesting they may have multiple mechanisms of action. The most potent tetracycline, methacycline, reduced the amount of Zika virus present in the brain and the severity of Zika virus-induced motor deficits in an immunocompetent mouse model. As Food and Drug Administration-approved drugs, the tetracyclines could be quickly translated to the clinic. The compounds identified through our screening paradigm have the potential to be used as prophylactics for patients traveling to endemic regions or for the treatment of the neurological complications of Zika virus infection.

Topics: Animals; Antiviral Agents; Artificial Intelligence; Chlorocebus aethiops; Disease Models, Animal; Drug Evaluation, Preclinical; High-Throughput Screening Assays; Immunocompetence; Inhibitory Concentration 50; Methacycline; Mice, Inbred C57BL; Protease Inhibitors; Quantitative Structure-Activity Relationship; Small Molecule Libraries; Vero Cells; Zika Virus; Zika Virus Infection

To investigate changes in free benzodiazepine receptor density in response to repeated, long-term administration of diazepam in epilepsy, we assessed 125I-iomazenil (125I-IMZ) binding in a mouse model.. El mice were divided into two groups of 12 mice each which received either no diazepam (E1(D[-]) group) or 2 mg/kg of diazepam per week (El(D[+]) group). Nine ddY mice were used as a control. Once each week from the age of 5 to 19 weeks, the El mice received stimulation to produce epileptic seizures 20 minutes after receiving intraperitoneal injections. At 20 weeks of age, a total dose of 0.37 MBq of 125I-IMZ was injected in all mice and their brains were rapidly removed 3 hours later. The incidence of epileptic seizures at the age of 19 weeks and the autoradiograms of the brain were compared.. The incidence of epileptic seizures in response to weekly stimulation was significantly lower in the E1(D[+]) group than in the E1(D[-]) group (p < 0.001). The percent injected doses of 125I-IMZ per gram of tissue in the cortex, hippocampus and amygdala were significantly lower in the E1(D[+]) group than in the E1(D[-]) group (p < 0.05).. The results suggest that diazepam binds competitively to 125I-IMZ as an agonist to free benzodiazepine receptor sites in the cortex, hippocampus and amygdala and shows anticonvulsant effect in E1 mice.

Topics: Animals; Anticonvulsants; Autoradiography; Brain; Diazepam; Disease Models, Animal; Epilepsy; Flumazenil; Iodine Radioisotopes; Mice; Protein Binding; Receptors, GABA-A; Seizures; Time Factors

To test the change in free or unoccupied benzodiazepine receptor (BZR) density in response to diazepam, we investigated (125)I-iomazenil ((125)I-IMZ) binding and serum corticosterone levels in a rat model. Wistar male rats, which received psychological stress using a communication box for 5 days, were divided into two groups according to the amount of administered diazepam: no diazepam [D (0)] group and 10 mg/kg per day [D (10)] group of 12 rats each. The standardized uptake value (SUV) of (125)I-IMZ of the D (10) group were significantly lower (P < .05) than those of the D (0) group in the frontal, parietal and temporal cortices, globus pallidus, hippocampus, amygdala and hypothalamus. The serum corticosterone level ratio in the D (10) group was significantly lower than that in the D (0) group (P < .05). From the change in serum corticosterone levels, diazepam attenuated the psychological stress produced by the physical stress to animals in adjacent compartments. From the reduced binding of (125)I-IMZ, it is clear that diazepam competed with endogenous ligand for the free BZR sites, and the frontal, parietal and temporal cortices, globus pallidus, hippocampus, amygdala and hypothalamus are important areas in which (125)I-IMZ binding is strongly affected by administration of diazepam.

Topics: Animals; Brain; Corticosterone; Diazepam; Disease Models, Animal; Flumazenil; Male; Radionuclide Imaging; Radiopharmaceuticals; Rats; Rats, Wistar; Receptors, GABA-A; Statistics as Topic; Stress, Psychological; Tissue Distribution

Gamma-aminobutyric acid (GABA)-A/benzodiazepine receptors (BZRs) play an important inhibitory role in epileptogenesis. [123I]Iomazenil (123I-IMZ) is a specific ligand for central-type (or neuronal-type) BNRs and is available for single-photon emission computed tomography (SPECT) in brain disorders. We demonstrated alterations of central-type BZRs in human focal epilepsies and their experimental models.. We examined interictal 123I-IMZ SPECT in patients with mesial temporal lobe epilepsy (MTLE; n = 19) with hippocampal sclerosis and neocortical epilepsy with focal cortical dysplasia (NE-CD; n = 18), and compared those with magnetic resonance imaging (MRI) and 123I-IMP SPECT (for regional cerebral blood flow). We also investigated in vitro autoradiography with (123)I-IMZ at various time courses in the intraamygdala kainate, amygdala kindling, and in-utero irradiation models.. In MTLE patients, the epileptogenic hippocampus often showed decreases in both 123I-IMZ and 123I-IMP SPECT. Consistent with those, marked reduction of 125I-IMZ binding was observed in hippocampal CA1-3 regions of the kainate model, which clearly paralleled pyramidal neuronal loss. In contrast, 125I-IMZ binding was increased in the dentate gyrus at 1 month but returned to the normal level at 3-6 months, when frequent spontaneous seizures appeared. The amygdala-kindling model demonstrated similar increases in 125I-IMZ binding in the dentate gyrus without any changes in other brain regions. In NE-CD patients, the epileptogenic foci showed decreased 123I-IMZ binding with relatively normal 123I-IMP SPECT. 125I-IMZ binding also was decreased in the cerebral cortex, hippocampus (areas CA1, 2, and 4), and caudate/putamen of the in-utero irradiation model.. These results indicate that central-type BZRs neuroimaging is useful for detection of epileptogenic foci, but their alterations differ between epilepsy subtypes and time-courses.

Topics: Adult; Amygdala; Animals; Autoradiography; Cerebral Cortex; Dentate Gyrus; Disease Models, Animal; Epilepsies, Partial; Epilepsy, Temporal Lobe; Female; Flumazenil; Hippocampus; Humans; Iodine Radioisotopes; Kainic Acid; Kindling, Neurologic; Magnetic Resonance Imaging; Male; Neocortex; Rats; Receptors, GABA-A; Regional Blood Flow; Tomography, Emission-Computed, Single-Photon

Iodine-123 labelled iomazenil ([(123)I]IMZ) has been reported to be a useful marker of neuronal viability. The brain distribution of [(123)I]IMZ, however, has not been correlated with the pathophysiological response in detail after an ischaemic insult. To characterise [(123)I]IMZ as a marker of neuronal viability, we compared its brain distribution with cyclooxygenase-2 (COX-2) expression, DNA fragmentation and cellular integrity. [(123)I]IMZ and [(125)I]IMP were injected into rats with focal cerebral ischaemia for the purpose of dual-tracer autoradiography. COX-2 and microtubule-associated protein-2 (MAP-2, a marker of cellular integrity) were immunostained. In situ DNA polymerase-I-dependent dUTP incorporation into damaged DNA was used as an indicator of DNA fragmentation. Lesion to normal ratios (LNRs) for [(123)I]IMP and [(125)I]IMZ were calculated. [(123)I]IMZ accumulation was preserved in several regions with impaired [(123)I]IMP accumulation. COX-2 expression was occasionally observed, whereas neither DNA fragmentation nor MAP-2 denaturation was detected in these regions. DNA fragmentation and impaired MAP-2 immunostaining were observed only in the regions with reduced LNRs for both tracers. The LNR for [(123)I]IMZ was significantly lower in regions with impaired MAP-2 immunostaining (0.120+/-0.152, P<0.0001), in regions positive for dUTP incorporation (0.488+/-0.166, P<0.0001) and in regions positive for COX-2 expression (0.626+/-0.186, P<0.001) than in histologically normal regions (0.784+/-0.213). Thus, neuronal DNA is still intact and cellular integrity is maintained in the ischaemic regions with preserved [(123)I]IMZ accumulation. The impairment of [(123)I]IMZ accumulation precedes DNA fragmentation and denaturation of cellular integrity. These results provide the molecular basis of [(123)I]IMZ distribution.

Topics: Animals; Brain Ischemia; Disease Models, Animal; Flumazenil; Iodine Radioisotopes; Male; Metabolic Clearance Rate; Radionuclide Imaging; Radiopharmaceuticals; Rats; Rats, Sprague-Dawley; Tissue Distribution

1-(11)C-Octanoate is a potential tracer for studying astroglial function in PET. To evaluate the usefulness of 1-(11)C-octanoate for studying ischemic stroke, we investigated the brain distribution of 1-(14)C-octanoate and compared it with N-isopropyl-p-(123)I-iodoamphetamine ((123)I-IMP) distribution (cerebral blood flow), (123)I-iomazenil ((123)I-IMZ) distribution (neuronal viability based on (123)I-IMZ binding to benzodiazepine receptors), and hematoxylin-eosin stain (morphologic changes) in a rat model of focal cerebral ischemia.. The right middle cerebral artery of each rat was occluded intraluminally. The brain distribution of 1-(14)C-octanoate and (123)I-IMP (or (123)I-IMZ) was determined 4 and 24 h after the insult using a dual-tracer autoradiographic technique (n = 4-7 in each group). Coronal brain sections adjacent to those used for autoradiography were stained with hematoxylin and eosin. Regions of interest (ROIs) were determined for 3 coronal slices, and asymmetry indices (AIs, lesion/normal hemisphere) of the tracer uptake were calculated. ROIs on the hemisphere with the lesion were classified into 4 groups: In region A, widespread necrotic cells were observed; in region B, necrotic cells were occasionally observed; in region C1, no morphologic changes were observed and the AIs for (123)I-IMP (or (123)I-IMZ) were 0.8.. 1-(14)C-Octanoate uptake decreased in the regions where morphologic changes were observed (regions A and B) but was relatively preserved in the surrounding region without morphologic changes despite reduced (123)I-IMP and (123)I-IMZ uptake (region C1). In the region without morphologic changes (region C1), AIs for 1-(14)C-octanoate were significantly higher than those for (123)I-IMP (4 h, 0.73 +/- 0.23 for 1-(14)C-octanoate and 0.37 +/- 0.20 for (123)I-IMP, P < 0.0001; 24 h, 0.84 +/- 0.11 for 1-(14)C-octanoate and 0.44 +/- 0.15 for (123)I-IMP, P < 0.0001) and those for (123)I-IMZ (4 h, 0.83 +/- 0.19 for 1-(14)C-octanoate and 0.57 +/- 0.13 for (123)I-IMZ, P < 0.0001; 24 h, 0.91 +/- 0.13 for 1-(14)C-octanoate and 0.73 +/- 0.06 for (123)I-IMZ, P < 0.0001).. 1-(14)C-Octanoate uptake was relatively preserved in the regions without morphologic changes despite reduced (123)I-IMP and (123)I-IMZ uptake. 1-(11)C-Octanoate may provide further functional information on the pathophysiology of ischemic stroke, reflecting astroglial function based on fatty acid metabolism.

Topics: Animals; Autoradiography; Brain; Brain Ischemia; Caprylates; Disease Models, Animal; Flumazenil; Iofetamine; Radiopharmaceuticals; Rats; Rats, Sprague-Dawley; Tissue Distribution

I-123-iomazenil is a SPECT probe for central benzodiazepine receptors (BZR) which may reflect intact cortical neuron density after ischemic insults. We evaluated whether neuronal damage in rats could be characterized by iomazenil as compared with cerebral blood flow (CBF). Serial changes in I-125-iomazenil for BZR and I-123-IMP for CBF were analyzed after the unilateral middle cerebral artery occlusion in rats by using an in vivo dualtracer technique. Uptake ratios of affected to contralateral regions were calculated. The iomazenil as well as IMP were decreased in all regions except for the cerebellum (remote area). Both iomazenil and IMP increased over time except in the temporal region (ischemic core). The iomazenil uptake was higher than IMP except in the ischemic core between 1 and 3-4 wk when iomazenil was lower than IMP. Iomazenil showed a moderate decrease in the proximal and middle parietal regions (peri-infarct areas) at 3-4 wk. The triphenyl-tetrazolium-chloride (TTC) stain at 1 wk demonstrated unstained tissue in the temporal region indicating tissue necrosis. With hematoxylin-eosin (HE) stain at 1 wk, widespread neuronal necrosis with occasional intact neurons were found in the proximal parietal region, and isolated necrotic neurons were represented in the distal parietal region. Iomazenil correlated well with the neuron distribution and the finding of a discrepancy between iomazenil and IMP might be useful in evaluating the neuronal damage.

Topics: Animals; Brain; Cerebrovascular Circulation; Disease Models, Animal; Flumazenil; Iodine Radioisotopes; Ischemic Attack, Transient; Male; Necrosis; Neurons; Rats; Rats, Sprague-Dawley; Time Factors; Tomography, Emission-Computed, Single-Photon