N-Methyl-N-Nitrosourea-Induced Acute Alteration of Retinal Function and Morphology in Monkeys.

PURPOSE: The purpose of this study was to investigate both functional and morphologic alteration of the retina acutely induced by N-methyl-N-nitrosourea (MNU) in monkeys. METHODS: The MNU was administered intravenously at a single dose of 40 mg/kg to six cynomolgus monkeys, and standard full-field electroretinograms (ERGs) were recorded 1, 3, and 7 days after dosing. In addition, the rod and cone a-waves in response to high-intensity flashes were analyzed by the a-wave fitting model (a-wave analysis). The photopic negative response (PhNR) was also recorded at the same time points. Furthermore, the retinas of two animals each were examined histopathologically 1, 3, or 7 days after dosing. RESULTS: The MNU attenuated all the standard full-field ERGs including the rod-driven and cone-driven responses; in the combined rod-cone response, the b-wave was more affected than the a-wave. In the a-wave analysis, the sensitivity parameters (S) of the rod and cone a-waves had decreased on the day after dosing and remained unchanged thereafter. The maximum response parameter (Rmax) of the rod a-wave gradually decreased. On the other hand, the Rmax in the cone a-wave transiently increased on the day after dosing and decreased thereafter; the PhNR amplitude showed a similar time course change. Histopathologically, the retinal lesion on the day after dosing mainly consisted of pyknosis and karyorrhexis in the photoreceptor nucleus. Depletion of some photoreceptor nuclei, and shortening and disorientation of the photoreceptor segments became prominent at 3 and 7 days after dosing. Localization of degenerated photoreceptors was consistent with that of rhodopsin-positive photoreceptors, resulting in a well-preserved central fovea. CONCLUSIONS: Our results indicated that MNU acutely induced rod-dominant photoreceptor degeneration in monkey retinas, but the photoreceptor function was impaired in both the rods and cones. Functional involvement of the postreceptoral components was also indicated.

Sildenafil-induced reversible impairment of rod and cone phototransduction in monkeys.

PURPOSE: To investigate functional alteration of the retina induced by sildenafil in monkeys. METHODS: Sildenafil was administered intravenously to cynomolgus monkeys at dose levels of 0, 1, 3, and 10 mg/kg, and standard full-field electroretinograms (ERGs) were recorded. The rod and cone a-waves in response to high-intensity flashes were also analyzed by the a-wave fitting model (a-wave analysis). Additionally, the photopic negative responses were recorded. RESULTS: Sildenafil at 3 mg/kg or more induced the following alterations in the standard full-field ERGs immediately after dosing: delayed b-wave in the rod response; delayed a-wave in the combined rod-cone response; and attenuated b-waves in the single-flash cone response and in the 30 Hz flicker. Additionally, the following changes were observed in the 10 mg/kg group: attenuated b-wave in the rod response; attenuated a-wave and delayed b-wave in the combined rod-cone response; delayed oscillatory potentials; and attenuated and delayed a-wave in the single-flash cone response. In the a-wave analysis immediately after dosing, sildenafil selectively decreased the sensitivity parameter (S) in the cone a-wave at 3 mg/kg, and in both the rod and cone a-waves at 10 mg/kg. The S value was highly correlated with plasma sildenafil concentration. The above changes fully recovered 24 hours after dosing. CONCLUSIONS: Sildenafil produced reversible impairment of the rod and cone phototransduction in monkeys. Meanwhile, involvement of the postreceptoral retinal components was suggested. These findings contribute to the clarification of sildenafil-induced visual disturbances. It is suggested that the photoreceptors are predominantly, but not exclusively, affected in the retina of humans with sildenafil-induced visual disturbances.

Digoxin-induced reversible dysfunction of the cone photoreceptors in monkeys.

PURPOSE: To investigate functional alteration of the retina induced by digoxin in monkeys. METHODS: Digoxin was intravenously administered to cynomolgus monkeys and standard full-field electroretinograms (ERGs) were serially recorded. In other digoxin-treated monkeys, the rod and cone a-waves to high-intensity flashes were obtained and analyzed by the a-wave fitting model (a-wave analysis). The following responses were also recorded: dark- and light-adapted responses to flashes of different intensities (dark- and light-adapted luminance responses), photopic ERG elicited by long-duration stimulus (ON-OFF response), and the photopic negative response (PhNR). RESULTS: Delayed b-wave was observed in all responses of the standard full-field ERGs; amplitude of the b-wave was increased in the rod response, but was decreased in the single-flash cone response and the 30-Hz flicker. These changes recovered gradually after elimination of digoxin from the blood. Digoxin enhanced and delayed the b-wave in the dark-adapted luminance-response analysis regardless of stimulus intensity. In the light-adapted luminance-response analysis, digoxin attenuated the a- and b-waves only at high and middle stimulus intensity, respectively. The a-wave analysis revealed selective decrease in the maximum response parameter (Rmax) in the cone a-wave. Both the b- and d-waves of the ON-OFF response were delayed. CONCLUSIONS: The selectively reduced Rmax in the cone a-wave indicated dysfunction of the cone photoreceptors in digoxin-treated monkeys. Meanwhile, the enhanced and delayed rod response suggested alteration of retinal components other than the cone photoreceptors. These results may contribute to the understanding of digoxin-induced visual disturbances in humans. It is suggested that the cone function is markedly, but not exclusively, affected in the retina of such patients.

Retinal function and morphology in monkeys with ethambutol-induced optic neuropathy.

PURPOSE: Ethambutol-induced optic neuropathy is a well recognized adverse ocular event. However, abnormalities of the retina in this optic neuropathy are not fully understood. Therefore, the purpose of the present study was to investigate both functional and morphological alterations of the retina induced by ethambutol in monkeys. METHODS: Ethambutol was orally administered to three cynomolgus monkeys, initially at 400 mg/kg/day followed by 800 mg/kg/day, for a maximum of 39 weeks. Full-field electroretinograms (ERGs) were recorded at intervals of approximately one month. The protocol included standard ERG responses to white flashes obtained under dark-adapted conditions (rod, combined rod-cone, oscillatory potentials) or with a white background (single-flash cone, 30 Hz flicker). In addition, we measured the ERG elicited with red flashes under blue background light (single-flash cone response [R/B]). All the ethambutol-treated monkeys were euthanized, and the retinae and various other nervous system tissues were examined histopathologically. RESULTS: No obvious changes were observed in the standard full-field ERGs. On the other hand, selective attenuation of the photopic negative response (PhNR) of the single-flash cone response (R/B) was observed in two out of three ethambutol-treated monkeys at week 22 or 28. Histopathology of these two monkeys revealed single cell necrosis of the retinal ganglion cells (RGCs), decreased RGCs in the parafovea and increased microglial cells in the nerve fiber layer in the retina, in addition to demyelination and glial reaction in the optic nerve, chiasm and tracts. CONCLUSIONS: The attenuated PhNR and histopathology of the retina indicated that RGCs were markedly damaged, both functionally and morphologically in monkeys with ethambutol-induced optic neuropathy. These results implied that RGCs are predominantly affected in the retina of patients with ethambutol-induced optic neuropathy.

Mechanism of voriconazole-induced transient visual disturbance: reversible dysfunction of retinal ON-bipolar cells in monkeys.

PURPOSE: To investigate the mechanism of voriconazole-induced transient visual disturbance in humans. METHODS: andard full-field electroretinograms (ERGs) were recorded from monkeys treated intravenously with voriconazole. In addition, photopic ERGs elicited by long-duration stimuli (ON-OFF response) were also recorded from monkeys receiving intravenous voriconazole or intravitreal 2-amino-4-phosphonobutyric acid (APB). RESULTS: aracteristic changes were observed in the waveform of the standard full-field ERGs obtained immediately after dosing of voriconazole as follows: electronegative combined rod-cone response (markedly attenuated b-wave and oscillatory potentials), undetectable rod response (eliminated b-wave); slightly abnormal single-flash cone response (flattened appearance in the bottom of the a-wave, mildly attenuated b-wave); and slightly abnormal 30 Hz flicker (mildly attenuated b-wave). The above changes fully recovered to baseline 24 hours after each dosing, along with a decrease in plasma voriconazole concentration. In addition, the change in the waveform of the ON-OFF response recorded in voriconazole-treated monkeys was quite similar to that recorded in APB-treated monkeys as follows: the b-wave was eliminated or prominently attenuated; and the a- and d-waves were not apparently attenuated. CONCLUSIONS: The results strongly suggest that voriconazole induces selective and reversible dysfunction of the retinal ON-bipolar cells in both the rod and cone pathways in monkeys. From the results obtained in monkeys in this study, it is suggested that the function of the retinal ON-bipolar cells was selectively and reversibly affected in voriconazole-treated humans who complained of transient visual disturbances.

Age-related differences of QT interval and autonomic nervous system activity in female cynomolgus monkeys.

INTRODUCTION: Cynomolgus monkeys are used in in vivo toxicological studies to evaluate the effects of drug candidates on the cardiovascular system, especially the effects of drugs on the QT interval on the electrocardiogram (ECG). Aging is reportedly one of the factors influencing the QT interval, but data from old monkeys have not been available. METHODS: The ECG parameters, including the QT interval and rate-corrected QT intervals calculated using Bazett's formula (QTcB) or individual correction factors (QTcI), in old female monkeys (the old group, n=7, average age=25.1+/-1.1 years) or young female monkeys (the young group, n=7, average age=4.4+/-0.2 years) were assessed by Holter electrocardiogram monitoring. The prolongation of QT interval induced by dl-sotalol, a representative class III antiarrhythmic drug, was also evaluated. In addition, power spectral analysis of heart rate variability was conducted. RESULTS: The QT interval in the old group was shorter than that of the young group during the dark period. The power spectral analysis of the ECG revealed quite a difference in autonomic nervous system activity between old and young animals. The prolongations of the QT interval, QTcB and QTcI after oral administration of 3 mg/kg dl-sotalol in the old group tended to be greater than those in the young group, and the QT interval in the old group was significantly longer than that in the young group at 1 h after dosing of dl-sotalol. DISCUSSION: The present study revealed some of the influences of age on the QT interval in female cynomolgus monkeys. There may be age-related differences in the circadian variation of QT interval or the drug-induced QT interval prolongation in this species.

Evaluation of drug-induced QT prolongation in a halothane-anesthetized monkey model: effects of sotalol.

INTRODUCTION: Cynomolgus monkeys are used in in vivo models of safety pharmacological studies to evaluate the effects of drug candidates on the cardiovascular system. Models using halothane-anesthetized animals have been used for the detection of drug-induced QT interval prolongation, but few studies with anesthetized monkeys have been reported. METHODS: The electrophysiological changes induced by dl-sotalol, a representative class III antiarrhythmic drug, were assessed in halothane-anesthetized monkeys (n = 4) or conscious and unrestrained monkeys (n = 4). RESULTS: In terms of basal characteristics, the QT interval was longer and the heart rate (HR) was lower under anesthesia than those under conscious conditions. Intravenous administration of 0.1 to 3 mg/kg dl-sotalol to anesthetized monkeys decreased the HR and prolonged the QT interval, monophasic action potential (MAP) duration and ventricular effective refractory period in a dose-dependent manner. In addition, reverse use-dependent prolongation of MAP duration was detected by electrical pacing, whereas the terminal repolarization period was hardly affected at any dose. Oral administration of 3 to 30 mg/kg dl-sotalol to conscious monkeys also decreased the HR and prolonged the QT interval in a dose-dependent manner. When compared at similar plasma concentrations of sotalol, the extent of QT interval prolongation under halothane anesthesia was equal to or greater than that under conscious conditions. DISCUSSION: The sensitivity for detection of drug-induced QT prolongation under halothane anesthesia may be satisfactory compared with that under conscious conditions. The present examinations indicated the usefulness of a model using halothane-anesthetized monkeys for evaluation of drug-induced QT interval prolongation.

Age-associated changes of flash visual evoked potentials in dogs.

Age-associated changes of visual evoked potentials by flash stimulation (flash VEP) were evaluated in 53 beagle dogs aged from 1- to 15-year-old. Among the components of flash VEP consisted of 3 positive (P1, P2 and P3) and 2 negative (N1 and N2) peaks by 150 msec, the latency of P2 and the later peaks (N2 and P3) were significantly delayed with aging. Both amplitudes of the P2-N2 and N2-P3 also showed a significant correlation with aging. The flash VEP is considered to be an available and useful technique to evaluate not only for visual pathway, but also some disturbance of neurological functions, like as those reported in demented human.

Changes of magnetic resonance imaging on the brain in beagle dogs with aging.

Age-associated changes of magnetic resonance imaging (MRI) on the brain were evaluated in 19 beagle dogs aged from 8-month- to 16-year-old. A significant correlation of the volume of lateral ventricle space was observed in the dogs with age advanced, however, no correlation was found between hippocampus size and the aging. The hypo-intensity areas on T2-weighted MRI were detected in globus pallidus and substantia nigra with a significant correlation of both intensity ratios to lateral ventricle with age advanced. These areas were coincided with the accumulation of iron in the slice of the brain with Perls' staining. In addition, hyper-intensity area, suggesting perivascular demyelination with fluid-filled space, was also observed in white matter surrounding the lateral ventricle on T2-weighted MRI. These results suggested that age-associated changes of T2-weighted MRI were developed in the dog brain, especially in globus pallidus, substantia nigra, and white matter surrounding lateral ventricle, like as those reported in the human brain.

Topographic analysis of flash visual evoked potentials in dogs.

Visual evoked potentials by flash stimulation (flash VEP) were analyzed in dogs using a topographic method. The flash VEP consisted of 3 positive (P1, P2 and P3) and 2 negative (N1 and N2) components by 150 msec after the flash stimuli. On the topographic mappings, a negative response area was observed in the frontal region of the scalp in the stimulated site followed by the shifting of the area to the contralateral frontal region and occipital region, during the first 100 msec. The negative response area in the frontal region in the stimulated site, contralateral frontal and temporal region, and occipital region were corresponded to N1, P2, and N2 on the flash VEP, respectively, according to their latencies. In the dogs with experimentally impaired the right lateral geniculate body, the latency of P2 was prolonged, and N2 and P3 were disappeared after the left eye stimulation. On the topographic mapping, only the early negative response area was detected on the stimulated site of the frontal region of the brain. Therefore, it is concluded that P1 and N1, P2, and N2 are referred to the retinal potentials, the potentials from the retina to the brainstem included the lateral geniculate body, and those from the brainstem to the visual cortex, respectively.

Subcutaneous continuous glucose monitoring and dose adjustment decreases glycosylated hemoglobin in spontaneously diabetic cynomolgus monkeys.

Spontaneously diabetic cynomolgus monkeys (Macaca fascicularis) exhibit a condition similar to human type 2 diabetes. These monkeys have been maintained by treatment with insulin therapy based on fasting blood glucose levels and glycosylated hemoglobin (HbA1c) values, which are determined periodically by blood sampling. Instead we sought to determine whether the MiniMed Continuous Glucose Monitoring System (CGMS, MiniMed Inc., Sylmer, Calif.), which takes glucose measurements continually over 24 h, would facilitate monitoring and treatment in diabetic cynomolgus monkeys. We attached the CGMS to five diabetic monkeys and obtained their blood glucose profiles. The performance of the CGMS was evaluated against blood glucose values obtained using a palm-sized blood glucose meter. The CGMS accurately measured the animals' blood glucose levels, with a median correlation of 0.95 and a mean absolute difference of 8.2% +/- 4.7% in comparison to the hand-held blood glucose meter. The diabetic monkeys were monitored two or three times during the 3-month study period. Throughout the study, the feeding time, dosage, and insulin administration time were changed in three of the five monkeys in light of the monitoring results. HbA1c levels were measured before and at 1 and 3 months after insulin adjustment. Although the adjusted dosage was not significantly different from the preceding one, HbA1c levels decreased from 7.6% +/- 1.3% to 6.5% +/- 1.1% (P < 0.05) by the end of the study. We concluded that the values from the CGMS closely correlated with those obtained with the hand-held blood glucose meter. Using the CGMS to determine blood glucose profiles allows the blood glucose levels of the monkeys to be monitored during the night as well as the day. Therefore, such continuous monitoring is useful in preventing nocturnal hypoglycemia and hypoglycemic seizures and may facilitate successful management of diabetes.

Effects of physical and chemical restraint on intravenous glucose tolerance test in crested black macaques (Macaca nigra).

The effects of physical and chemical restraint on glucose clearance and insulin secretion were evaluated during intravenous glucose tolerance testing in Macaca nigra. Conscious monkeys placed in plexiglas cylindrical restraining devices (CRD) appeared relaxed, but glucose clearance and insulin secretion were impaired. A combination of midazolam with ketamine, compared to ketamine alone, did not cause detectable changes in the intravenous glucose tolerance tests; midazolam also reduced adverse reactions to ketamine and extended the duration of anesthesia. The cylindrical restraining device can be convenient for examining monkeys, but it is limited by its adverse affects on metabolic and hormonal measurements in intravenous glucose-tolerance tests. Chemical restraint using ketamine with midazolam was more effective than ketamine alone.