Cell culture has become a routine laboratory technique. In recent years, many materials for cell culture have become commercially available. Cells are grown and maintained at an appropriate temperature and gas mixture (typically, 37°C, 5% CO2) in a cell incubator. Culture conditions vary widely for each cell type, and variation of the conditions for a particular cell type can result in the expression of different phenotypes. Some cells naturally live without attaching to a surface, such as cells that exist in the bloodstream. Others require a surface, such as most of the cells derived from solid tissues. Cells growing unattached to a surface are referred to as suspension cultures. Adherent cultured cells can be grown on tissue culture plastic, which may be coated with extracellular matrix components (e.g. collagen or fibronectin) to increase the cellular adhesion properties and provide other signals needed for growth. As cells generally continue to divide in culture, they generally grow to fill the available area or volume. Cell-to-cell contact can stimulate cell cycle arrest, causing the cells to stop dividing, known as contact inhibition. Cell-to-cell contact can stimulate promiscuous and unwanted cellular differentiation. These issues can be dealt with using tissue culture methods that rely on sterile techniques. In this paper, several kinds of cell culture methods and inner ear tissue culture are introduced.
Nystagmus is an important clinical finding in patients with neurologic and neuro-otologic diseases. However, the clinical significance of nystagmus has not yet been well evaluated in psychiatric patients with functional/organic brain dysfunction. In the present study, we performed gaze, positional and positioning nystagmus tests in 144 patients with psychiatric diseases (101 men; 61.0±13.0 years) in order to evaluate the frequency and characteristics of nystagmus in these patients. Patients were classified according to the underlying diseases. Nystagmus was seen in 41 (28.5%) out of the 144 cases. Nystagmus was seen in 15 (62.5%) of the 24 cases of alcoholism, 9 (27.3%) of the 33 with organic psychiatric disorders, 16 (20.5%) of the 78 with schizophrenia, 1 (25.0%) out of the 4 with mental retardation, 0 (0.0%) out of the 4 with mood disorders, and 0 (0.0%) out of the 1 with anxiety disorders. These results suggest that nystagmus may be a very important clinical finding not only in patients with neurologic and neuro-otologic diseases, but also in patients with psychiatric diseases.
Intratympanic instillation of gentamicin is currently a well-established treatment for intractable vertigo in patients with Meniere's disease. There are, however, only a few reports of this therapy in patients with non-Meniere type of vertigo. We report the case of a 68-year-old woman with the lateral semicircular canal type of benign paroxysmal positional vertigo (BPPV) following right idiopathic sudden hearing loss. The patient was annoyed by the positional vertigo for one and a half years and was successfully treated by intratympanic gentamicin instillation. She had profound hearing loss on the right side and direction-changing apogeotropic positional nystagmus. Her caloric response on the right side was severely decreased (canal paresis 80%). Intratympanic gentamicin instillation therapy was undertaken (30mg/ml/day × 5 days), following which her positional vertigo was abolished. We propose that intratympanic instillation of gentamicin may also be applicable to selected patients with long-lasting BPPV.
Benign paroxysmal positional vertigo (BPPV) is usually idiopathic, but in some cases, it arises after inner ear disease. We examined the cases with and without BPPV-like symptoms in sudden deafness (SD), Ménière's disease (MD) and vestibular neuritis (VN). The equilibrium function tests (caloric test, VEMP and SVV) results were not different between the groups with or without BPPV-like symptoms, but hospitalization was needed more frequently in cases with BPPV-like symptoms. The posterior semicircular canal (PSC) is the most frequent focus of both idiopathic BPPV and BPPV-like symptoms after inner ear disease. The recovery period from vertigo was longer in the cases with BPPV-like symptoms. The effect of physical therapy for the PSC type was equally favorable in cases with idiopathic BPPV and those with inner ear disease associated with BPPV-like symptoms. For the lateral semicircular canal (LSC) type, the effect was less pronounced in cases with idiopathic BPPV. Occurrence of BPPV-like symptoms associated with inner ear disease is possibly caused by the canalolithiasis induced by utricular disturbances.
Many studies have shown that optokinetic stimulation induces postural sway and that vision, in addition to vestibular and somatosensory inputs, plays an important role in postural control. In the present study, we examined individual differences in the postural sway induced by movement of visual images back and forth and the effect of the response to the projected image. Three kinds of visual images were used: "normal direction" (image moving backward), "reverse direction" (image moving forward), and "static" images. Subjects received visual stimulation via a head-mounted display while standing on a stabilometer with their feet aligned in the Romberg position, and the postural sway was measured. The subjects initially maintained the standing position at rest for one minute (a static image was presented) and were then given visual stimulation for two minutes, followed by a 2-minute presentation of the static image. The period of visual stimulation is referred to as "during stimulation" and the 2-minute period of presentation of the static image is referred to as "after stimulation". The postural sway was measured over time. In Experiment 1, the measurement was conducted 5 times under each stimulation condition. In many subjects, postural sway was induced in the direction of visual stimulation "during stimulation" and in the opposite direction "after stimulation" (normal type); however, some subjects showed the opposite postural sway (reverse type) or showed no induced postural sway at all (poor response type). Most subjects showed similar changes for each of the five stimulations. Individual differences in postural sway were considered to be induced by the effect of vision on the postural control system. In Experiment 2, the measurement was conducted using the same visual stimulation under two kinds of mental states: a sub-conscious condition, in which the subjects were instructed to watch the image without any assumption regarding their own motion, and a conscious condition, in which the subjects were instructed to assume that that they were moving in response to the projected image (although no actual walking motion occurred). Many subjects showed postural sway in the opposite direction when the state was changed, despite the image remaining the same. These results suggest that posture is smoothly controlled using visual information to allow adaptation to various environments.
The functions of the eighth cranial nerve system changes markedly during the early postnatal period. The vestibulo-ocular reflex appears on postnatal day 2 in rats and its gain increases rapidly within 10 days. During the same period, the proportion of the regularly firing vestibular neurons increases, while that of the irregularly firing neurons decreases. The intrinsic firing properties of the isolated vestibular ganglion cells are heterogeneous, and the low-voltage activated K+ channels are critical to determining the pattern of the spike discharges. The firing patterns of these cells show considerable changes during the first 14 days of postnatal life. This developmental change might be regulated by some neurotrophic factors. At the brainstem auditory synapses, the Ca2+ channel types that trigger transmitter release switch from a mixture of P/Q-, N-, and R-types to predominantly the P/Q-types. This change is caused by replacement of the N-and R-type Ca2+ channels by P/Q-type calcium channels at the presynaptic nerve terminals. Since P/Q-type Ca2+ channels are more efficient for transmitter release, the developmental change of the Ca2+ channel types may contribute to high-fidelity synaptic transmission. Synaptic efficacy also changes during the same postnatal period at this synapse. The size of the readily releasable pool of synaptic vesicles increases, while the release probability concomitantly decreases. These developmental changes contribute to the formation of stable synapses at which only a small fraction of releasable synaptic vesicles is depleted during high-frequency tranmission. The eighth cranial nerve system changes its firing properties, ion channels and synaptic efficacy during the early postnatal period, towards establishing efficient synaptic function.
Human perceptual-motor behaviors emerge from dynamic interaction among the neural system, musculoskeletal system and the environment. Generation of stable and flexible behaviors is captured on the basis of the nonlinear dynamical systems theory. The theory provides deep insight into the developmental mechanism of perceptual-motor behaviors in young infants. First, I shall present my argument that the spontaneous dynamics of the neuromuscular system in young infants is the key for the acquisition of new actions in the complex environment. Second, I discuss the mechanisms of selection of frames of reference, such as the retinal and environmental coordinates, and of the integration of different frames of reference in young infants. Some of the empirical studies addressing these issues are shown.
The clinical application of transcranial magnetic stimulation (TMS) for prognostic evaluation of peripheral facial palsy is discussed. Because the recovery of motor nerve function is determined by the degree of wallerian degeneration and degenerated nerve fibers are not responsive to electrical stimulation, examinations using electrical stimulation are important for the accurate prognostic evaluation of peripheral facial palsy. However, when the facial nerve is stimulated peripheral to the injury site, the prognosis cannot be estimated correctly until the wallerian degeneration is complete at the site of stimulation. Thus, the results of the nerve excitability test (NET) and electroneurography (ENoG) are not significant for the prognostic evaluation until 7-10 days after the onset of the palsy. When the coil of the magnetic stimulator is placed on the temporal portion at a point 6cm above and 3cm behind the external auditory meatus, the facial nerve can be stimulated around the fundus of the internal auditory meatus, which is the central site of the injury in most cases of facial palsy. In normal subjects, the magnetically evoked compound muscle action potential (CMAP) recorded from the electrodes placed on the orbicularis oris muscle is similar to the CMAP in ENoG. But the latency of the CMAP evoked by TMS delays about 1 msec compairing with the CMAP in ENoG. Although the CMAP evoked by TMS can be recorded in about a half of the patients and the amplitude is very low in the early stages of palsy, 83% of the patients showing a response evoked by TMS in the early stages show good prognosis. Patients with a good prognosis can thus be identified using TMS even in the early stages of palsy. The sensitivities and specificities of the examinations used for prognostic evaluation, such as the scoring system for facial movements, NET, stapedial reflex and TMS, are also discussed.
By means of noisy galvanic vestibular stimulation (GVS), it might be possible to ameliorate the blunted responsiveness of degenerated neuronal circuits in patients with degenerative neurological diseases. We evaluated the effects of 24-hour noisy GVS on the long-term heart rate dynamics in patients with multiple system atrophy and on the daytime trunk activity dynamics in patients with either levodopa-responsive Parkinson's disease or levodopa-unresponsive parkinsonism patients. Patients were also examined for cognitive performance by means of a continuous performance test. Short-range or high-frequency fluctuations of the heart rate were significantly increased by the noisy GVS as compared with that by sham stimulation, suggestive of improved autonomic, especially parasympathetic, responsiveness. The long-range anti-persistency of trunk activity patterns probed by an autocorrelation measure was significantly increased by the noisy GVS, suggestive of quickening of bradykinesic rest-to-active transitions. The mean reaction time in the continuous performance test was also significantly decreased by the noisy GVS, without significant changes in either the omission or commission error ratios, which is suggestive of improved motor execution during the cognitive task. Thus, noisy GVS improved the motor and autonomic responsiveness and is effective for ameliorating the symptoms in patients with multiple system atrophy or Parkinson's disease.
In an attempt to control hypokinetic and hyperkinetic movement disorders, deep brain stimulation (DBS) has been developed during the last two decades by several investigators. In 1987, Benabid and his colleagues suggested the usefulness of high-frequency stimulation of the ventral intermediate nucleus of the thalamus for treating drug-resistant tremors and avoiding the adverse effects of thalamotomy. Since then, DBS has been used as an alternative to functional neurosurgery for movement disorders, and more recently, it has been applied to the treatment of epilepsy, obsessive-compulsive disorders and cluster headache, in addition to other applications in experimental models. In regard to the treatment of movement disorders, recent clinical studies have demonstrated that DBS affords great benefits in terms of improvement of the activities of daily living in patients with Parkinson's disease (PD), essential tremor, dystonia and poststroke hyperkinetic movement disorders. We have treated patients with movement disorders by DBS of the thalamic nuclei ventralis oralis (Voa/Vop) et intermedius (Vim), globus pallidus internus (GPi), and subthalamic nucleus (STN). The site of permanent electrode placement was identified using magnetic resonance imaging and multiunit extracellular recording. The implantable pulse generator was internalized after postoperative test stimulation for one week. The stimulation parameters were modified by physicians at each follow-up visit on the basis of the findings on neurological examination, as well as the patient's report concerning the activities of daily living. The advantages of DBS include reversibility and controllability of stimulation. In addition, DBS carries a smaller risk of side effects, particularly when employed bilaterally. Thalamic DBS is useful for controlling tremor that is unresponsive to medication. DBS of the STN and GPi improves the motor functions in PD patients, mainly during the off-period. Moreover, STN-DBS attenuates levodopa-induced dyskinesia through reducing the requirement of DOPA, whereas GPi-DBS directly attenuates DOPA-induced dyskinesia. In addition, GPi-DBS is very useful for controlling the symptoms of idiopatic generalized dystonia. According to reports, DBS is associated with few serious adverse effects associated with DBS. In general, the operative mortality is less than 1%. The incidences of hemorrhage are in the range of about 1-6%, and the incidences of device-related complications, such as infection or skin erosion, are in the range of 3-26%. DBS is clinically effective in well-selected patients and should be considered as a treatment option for patients with medically refractory movement disorders. Despite its clinical usefulness, the mechanism underlying the efficacy of DBS is still unclear. There is no proof currently that long-term DBS can reset neural networks or induce profound modifications of functional organization. Several researchers have proposed hypotheses concerning the mechanism underlying the efficacy of DBS, including 1) jamming of neural transmission, 2) direct inhibition of spike initiation at the level of the membrane that may be due to the activation of inhibitory terminals, 3) functional changes due to a decrease or increase in the amount of neurotransmitter released, and 4) retrograde activation of upstream neural structures. From the viewpoint of basic neuroscience, the development of DBS is intriguing. Investigation regarding the mechanism underlying the efficacy of DBS may provide clues for further clarification of various processes in the central nervous system.
We report on the combined application of acoustic VEMP and electric VEMP for the diagnosis of vestibular dysfunction. For the recording of the acoustic VEMP, we used click (0.1msec, 95dBnHL) and 500-Hz short tone burst (rise/fall time 1msec plateau time 2msec, 95dBnHL) stimuli. For the recording of the electric VEMP, we used 3-mA (1msec) electrical stimulation. In the first part of the study, we applied this combined stimulation to patients who showed absent VEMP to click stimulation. Patients with labyrinthine lesions such as Meniere's disease showed normal electrical VEMP, although they showed no response to click stimulation. On the other hand, almost all patients with nerve lesions such as acoustic neuroma showed absent or decreased VEMP to electrical stimulation. These results suggest that electrical VEMP in patients with absent click-VEMP could be useful for the differentiation of labyrinthine lesions from nerve lesions. We applied this testing method to patients with vestibular neuritis and Ramsay Hunt syndrome. Patients with vestibular neuritis and Ramsay Hunt syndrome were classified in 2 groups. The majority had nerve lesions, while some patients had labyrinthine lesions. In the second part of the study, we applied the combined stimulation to patients with preserved acoustic VEMP. Then we calculated the acoustic-electric ratio of VEMP. In comparison with that in healthy subjects, patients with acoustic neuroma, and the unaffected sides of patients with Meniere's disease, the affected sides in patients with Meniere's disease showed a smaller acoustic-electric ratio of VEMP. These findings suggest hat the combined application of electrical VEMP and acoustic VEMP could be useful in the differentiation of labyrinthine lesions from nerve lesions even in patients with preserved acoustic VEMP.