Detection of mitochondrial DNA nucleotide 11778 point mutation of Leber hereditary optic neuropathy from archival stained histopathological preparations.

PURPOSE: We evaluated the availability of archival histopathological preparations for genetic diagnosis of Leber hereditary optic neuropathy (LHON). METHODS: Preparations of various tissues of an autopsied case of LHON, and of the optochiasmal arachnoidea of nine cases of bilateral optic neuropathy (BON) were studied to determine the presence of a point mutation of the mitochondrial DNA nucleotide (nt) 11778 using PCR method. RESULTS: An nt11778 point mutation was detected in all preparations of the autopsied case. Five preparations out of six BON cases who were diagnosed as LHON based on positive family history, revealed this point mutation. This mutation was also detected in two of three BON patients with no family history of the disease. CONCLUSION: The archival preparations were found to be available as materials of genetic diagnosis for LHON, which indicated that it would be capable to reevaluate retrospectively the pedigree of LHON and BON cases.

Vasomotor nerves of vessels in the human optic nerve.

Aminergic and cholinergic vasomotor nerves in vessels of the human optic nerve were studied morphologically. Aminergic nerve fibers were observed by the glyoxylic acid method. Cholinergic nerve fibers were observed by light microscopy after acetylcholinesterase staining by the Karnovsky-Roots method and Tago's modified method. In the retrobulbar optic nerve behind the bulbus, aminergic and cholinergic vasomotor nerves were observed to be dense in the central retinal artery and vein and posterior ciliary arteries. A large number of vasomotor nerves were also demonstrated in vessels in the septum of the optic nerve, but they were sparse in pial vessels. Further centrally, a few vasomotor nerves were found in pial vessels of the intracanalicular and intracranial optic nerve, but few were observed in the septum of the optic nerve. At the optic chiasm they were densely distributed in pial vessels.

[A case of suprasellar arachnoid cyst followed up for a long time].

We followed a case of suprasellar arachnoid cyst for 12 years. The patient was a sixteen-year-old girl without particular problems in her general condition. She showed optic atrophy in both eyes and optic nerve hypoplasia with an inferotemporal quandranopsia in the left eye. A suprasellar arachnoid cyst communicating with the tubarachnoid space was found to extend into the sella turcica as an empty sella. A cyst wall was resected and a cyst-peritoneal shunt performed. After 12 years from the operation, sensitivity was slightly depressed in the visual field where it had already been disturbed. Although there are few reports in the literature on involvement of the optic nerves and chiasma by suprasellar arachnoid cysts, papilledema and optic atrophy are often found in children, and infero-temporal quandranopsia or homonymous hemianopsia have been reported. Visual field defects were most likely caused by compression of the optic nerve by cyst or prolonged papilledema. We also suspect that some kind of disturbance to the optic nerve occurred during extension of the arachnoid cyst as an empty sella, or during formation of arachnoid cyst in the fetus stage.

A case-control study on risk factors for leukemia in a district of Japan.

A case control study was performed with 142 leukemia patients and 284 controls matched for age and sex. Occupation, birth order, past medical history, and drinking and smoking habits were compared in these two groups. Persons born first or fourth were found to have a higher incidence of leukemia. History of a fracture was one of the risk factors for acute leukemia, and a history of gastroduodenal ulcer was a risk factor for chronic leukemia. This may suggest that extensive exposure to X-rays in diagnosis and treatment is a risk factor for leukemia. There was a significant dose-response relationship between the amount of smoking and the incidence of acute nonlymphocytic leukemia, but not between the amount of alcohol consumption and the incidence of leukemia. Thus, smoking was one of the risk factors for acute leukemia.

[The distribution of actin on cultured oligodendrocytes from rat optic nerve].

Two types of cultured oligodendrocytes (OLs) from rat optic nerve were analyzed for the distribution of actin, a major contractile protein, using immunocytochemistry. Type 1 OLs showed extensive network of processes and type 2 OLs showed elaborate membranous expansion along an extensive network. Actin was diffusely stained on an extensive network of processes in type 1 OLs and on the distal portion of membranous expansion in type 2 OLs, but some oligodendrocytes were not stained. It was demonstrated that the distribution of actin in type 1 OLs and type 2 OLs varied with development in oligodendrocyte differentiation and maturation. Our results suggest that undifferentiated and immature oligodendrocytes display remarkable cell movement to search for target axons, and differentiated and mature oligodendrocytes do not require cell movement when myelination has finished. In conclusion, it is considered that actin plays an important role in myelinogenesis.

The isolation of cultured oligodendrocytes from rat optic nerve.

Oligodendrocytes, the myelin-forming cells of the central nervous system, were cultured from newborn rat optic nerve employing a modified method of McCarthy and DeVellis, and cultured oligodendrocytes were analyzed for characteristic morphology and maturation events by phase contrast, MBP immunofluorescent and scanning electron microscopy. Two types of oligodendrocytes, type 1 OLs and type 2 OLs, on poly-L-lysine-coated dishes were obtained. Type 1 OLs showed extensive networks of processes and type 2 OLs elaborated membranous expansion along extensive networks, and it was demonstrated that type 2 OLs developed from type 1 OLs by phase contrast-microscopic observation. By scanning electron-microscopic observation, membrane structure in type 2 OLs clearly corresponded to membranous expansion. Our results suggest that type 1 OLs and type 2 OLs are in the different states of the myelinogenesis, and that membranous expansion and membrane structure in type 2 OLs correspond to myelin membrane.

[Responses in extraocular muscles from magnetic stimulation of the cat brain].

In 12 adult cats, pulsed magnetic stimuli were applied to the scalp, with the center of the stimulating coil over the interparietal bone. Recordings were obtained from the superior rectus, lateral rectus and inferior oblique muscles with concentric needle electrodes. In all cats, single muscle action potentials were recorded easily in all the muscles examined with latencies of 3.2-37.5 ms (mean 15.6ms). These responses disappeared after intravenous administration of pancuronium bromide. Furthermore, the elicited action potentials in the inferior oblique muscles disappeared by amputation of the inferior oblique branch of the oculomotor nerve. In some motor units, stronger stimuli shortened the latency of the responses. In most units, however, stimulus intensity did not influence the latency of responses over a wide range. The mean latency (15.6ms) is coincident with the latency of cortical spike potentials preceding voluntary saccades. This remarkable coincidence suggests that our pulsed magnetic stimuli have a very strong possibility of generation of human saccades. Thus, magnetic stimulation of the scalp may provide further information relevant to the normal operation of the oculomotor system.

Cervical afferent pathway to inferior oblique motoneuron in the cat.

The neuronal pathway implicated in the vertical cervico-ocular reflex (COR) was investigated electrophysiologically in chloralose anesthetized cats. The effect of bilateral C2 dorsal root afferent stimulation on inferior oblique motoneurons (IO-MN) was investigated by intracellular recording. Control disynaptic excitatory postsynaptic potentials elicited in IO-MNs following stimulation of the contralateral anterior semicircular canal nerve (ACN) were invariably facilitated by conditioning stimulation to both ipsilateral and contralateral C2 dorsal roots (DR) in all motoneurons tested. This result indicates that inputs from the C2 DR of both sides and the contralateral ACN converge onto secondary vestibular neurons ('common interneurons') which project directly to the IO-MN. Common interneurons mediating vestibular and cervical excitation to the IO-MNs were studied in the vestibular nuclei on the side opposite to the motoneurons by extracellular recording. Nineteen vestibular neurons were identified as common interneurons; they were distributed in the caudal half of the lateral nucleus and the rostral half of the descending nucleus. The present experiment provides electrophysiological evidence of the projection of upper cervical afferents to the ipsilateral vestibular nuclei. The difference in neuronal organization between the horizontal and vertical COR is also briefly discussed.

Histochemical studies of mitochondrial activities of cultured corneal endothelial cells of cat during wound-healing.

The corneal endothelial cells of the cat were cultured, and in 3 weeks the cells became confluent and formed a cell sheet. The cells were stained with rhodamine 123 and nitroblue tetrazolium (NBT). The frequencies of cells stained by these two methods changed during the 3-week culture, but at the end of the culture the frequencies were stabilized at 58 +/- 3.9 (mean +/- SD) % for the rhodamine 123 staining method and 35 +/- 3.7% for the NBT method. An oval wound of 600 X 400 microns was made in the center of the cultured endothelial cell sheet, and the cell migration was observed by phase contrast microscopy. Six hours after the wounding, the cells began to migrate toward the center of the wounded area, and in 24 hours the denuded area was almost completely covered by migrated cells. In 48 hours the wound was tightly covered by the migrated cells. One hour after the wounding, the cells in the wound margin were strongly stained by rhodamine 123, but not by NBT. The cultured cell sheet was divided into 4 concentric areas, ie, S1 was the center of the wound, S2 was the 100-microns-wide zone inside the original wound edge, S3 was the 100-microns-wide zone outside the original wound edge, and S4 was the 100-microns-wide zone outside the S3 zone. Topographical changes in rhodamine- and NBT-stained cells were observed over a 48-hour period. Rhodamine-positive cells increased in the S1 area, while a decrease occurred in the S2 and S3 areas. NBT-positive cells peaked at 12 hours after the wounding and decreased thereafter. It was concluded that the endothelial cells migrating to cover the wounded area showed a significant enhancement of mitochondrial activity, as indicated by the rhodamine 123 staining, but the succinic dehydrogenase activity revealed by NBT staining was rather suppressed.

Effects of spinal cord compression on repetitive impulse conduction of the ascending fibers of the dorsal column.

The effects of spinal cord compression on conduction of compound action potentials (CAPs) in the dorsal column fibers at various stimulus frequencies were analysed in pentobarbital anesthetised cats. The L 6 posterior root was given 1 to 500 Hz stimuli, and the CAPs were recorded from the L 2 posterior column. The 100th CAP was recorded at 100, 200 and 500 Hz. The L 4 cord segment was compressed by stepped increments (0.5 mm/5 min.) with a plastic plate (4 x 10 mm) until the responses at 1 Hz disappeared, then the compression was released. Before compression, each CAP at all frequencies showed almost uniform amplitudes and latencies. During compression, the CAPs were not altered significantly at any frequency until the spinal cord was compressed 2.5-4.0 mm. Further compression produced a progressive decrease in amplitude and increase in latency of the CAPs. At 500 Hz, however, the conduction block (amplitude loss, latency increase) was much severer than that produced at lower frequencies. After release of compression, the CAPs at all frequencies recovered progressively, and became almost stable after about 1 hour. The recovery rate of amplitude was only about 30% compared to the amplitude before compression at 500 Hz, and about 50% at 1 and 200 Hz. However, the recovery rate of amplitude at 100 Hz (65%) exceeded those at 1 and 200 Hz. To observe the difference in recovery rates among them, all the trains of CAPs were analyzed. Before compression, the amplitudes were almost uniform at stimulus frequencies as high as 500 Hz.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of visual deprivation on the visual and oculomotor system: acetylcholinesterase activity in oculomotor neurons.

The effects of visual deprivation (dark-rearing) on neurons in both the visual and oculomotor systems of black mice were studied using morphologic and histochemical techniques. In the neurons of the dorsal nucleus of the lateral geniculate body, the cytoplasm of dark-reared mice was less developed and the cytoplasm/nucleus ratio was significantly smaller in the dark-reared mice than in the controls. In contrast, large motoneurons in the oculomotor nucleus did not show any ultrastructural changes and the cytoplasm/nucleus ratio was normal. However, in large motoneurons of dark-reared mice, acetylcholinesterase (AChE)-positive areas were scattered in the cytoplasm, and the ratio of the AChE-positive areas to the cytoplasm area was significantly smaller than that in the controls.

Monoclonal antibody RG1 against rat Müller cells recognizes optic nerve and cerebral cortex glia in culture.

A monoclonal antibody designated as RG1 was produced by the fusion of mouse myeloma cells with spleen cells from mice immunized with rat retinas. The monoclonal antibody was shown to bind to Müller cells of the rat retina by the avidin-biotin peroxidase complex method. The screening of antibody-producing hybridomas and the analysis of the monoclonal antibody obtained were performed by enzyme-linked immunosorbent assay. The optic nerve, cerebral cortex and dorsal root ganglia were cultured from 17-day embryonic rats and maintained for 17-20 days. Expressions of the RG1 antigen were examined by indirect immunofluorescence in the cultured glial cells. In both the optic nerve and the cerebral cortex glial cells the RG1 antigen was expressed; however, the RG1 antigen was not expressed in cultured Schwann cells throughout the whole culture period. Our results suggest that the RG1 antigen is a common antigen in the supporting neuroectodermal glial cells in the central nervous system.

Common interneurones mediating cortical and tectal excitation of abducens motoneurones in the cat.

Tectal and cortical effects on abducens motoneurones were examined with intracellular recording techniques in cats under chloralose anaesthesia. Abducens motoneurones exhibited disynaptic EPSPs after stimulation of the contralateral superior colliculus and cerebral peduncle. The tectal disynaptic EPSPs were observed invariably in all motoneurones tested, while the peduncular EPSPs were observed only in 40% of motoneurones after stimulation of the contralateral cerebral peduncle. However, the tectal disynaptic EPSPs were consistently facilitated by conditioning peduncular stimulation in all motoneurones tested. These results indicated that the disynaptic excitatory tecto-abducens and cortico-abducens pathways shared common premotor interneurones. The common interneurones which mediated the tectal and cortical disynaptic excitation of abducens motoneurones were explored in the pons. These interneurones were identified by the criteria that they were fired monosynaptically from both the tectum and the cerebral peduncle and were activated antidromically from the abducens nucleus. Systematic threshold mapping for the antidromic activation in and around the abducens nucleus indicated that they gave off many collateral branches in the nucleus. Such neurones were found in the nucleus reticularis pontis caudalis, being distributed in the area extending 0.8-3 mm rostral to the rostral pole of the abducens nucleus, 1.3-2.7 mm deep from the dorsal surface of the brain stem, and 0.8-1.8 mm lateral from the midline. The present experiments strongly suggest that a group of neurones in the paramedian pontine reticular formation make direct excitatory connexions with abducens motoneurones and play a role of common interneurones that transmit both tectal and cortical commands.

Conductivity of dorsal column fibers during experimental spinal cord compression and after decompression at various stimulus frequencies.

The effects of spinal cord compression on conduction of dorsal column fibers at various stimulus frequencies were analyzed in pentobarbital anesthetized cats. The responses to L6 dorsal root stimulation at 1 to 500 Hz were recorded from the L2 cord dorsum. The L4 cord segment was compressed gradually until the compound action potential (CAP) at 1 Hz was flat. There was no significant change of CAP at any frequency during the first part of compression, but there was progressive conduction failure, which was more severe with increased stimulus frequency, at a later stage. After decompression, the CAPs at all frequencies recovered progressively for 1 hour but slowly thereafter. However, marked differences were observed in recovery rate at different stimulus frequencies. The recovery rate at 500 Hz was much slower than that at 1 Hz, whereas the recovery rate at 100 Hz exceeded those at 1 Hz. Serial analysis of a train of high frequency impulses revealed the following different response patterns with stimulus frequencies after decompression. At 333-500 Hz the amplitude of CAPs decreased progressively, whereas at 33-125 Hz it increased up to 110-134% of the first CAP and then reached an almost steady level. At 200-250 Hz the amplitude increased transiently and then decreased progressively. The latency increased with decreased amplitude, and decreased with increased amplitude. Conduction failure at a high stimulus frequency (500 Hz) was observed at the compression site. In contrast, augmentation of CAPs at moderately high stimulus frequency (100 Hz) was observed rostral to the compressing site. The conduction failure at high stimulus frequency indicates incomplete impairment of spike generation in axons injured by mechanical compression and that these axons can transmit impulses at a low stimulus frequency. High frequency stimulation may be useful for monitoring of the function of the CNS axons. The mechanism underlying the augmentation of CAPs at moderately high stimulus frequency is briefly discussed.

Single fiber electromyography in cat extraocular muscles.

Single fiber electromyography (SFEMG) was carried out in the extraocular muscles (EOMs) of 40 adult cats, and the recordings were analyzed. The small fluctuations of the interpotential interval (IPI), so-called "jitter phenomenon", were represented quantitatively as a mean consecutive difference (MCD). The MCD and the IPI recorded from the orbital layer of the rectus muscles were similar among the 4 rectus muscles: the MCD and the mean IPI measured in 58 motor units from the 4 rectus muscles averaged 25.82 microseconds and 0.94 ms, respectively. These values were similar to those recorded from the oblique muscles. The MCD and IPI were 34.12 microseconds and 1.70 ms, respectively, in the retractor bulbi muscles and 45.54 microseconds and 1.23 ms, respectively, in the triceps muscles. The MCDs of the rectus and inferior oblique muscles were considerably smaller than those in the retractor bulbi and the skeletal muscles.