[Perspectives of genome editing in otorhinolaryngology]. / Perspektiven der Genomeditierung in der Hals-Nasen-Ohren-Heilkunde.

BACKGROUND: Recent advances in DNA sequencing technology have enabled researchers to identify the genetic background underlying human illness. In addition, the latest genome editing technology, CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9), provides great potential to edit genomic DNA sequences precisely with high efficiency. This technology has been evaluated for treatment of genetic diseases in recently published preclinical studies. Since many such genetic disorders can affect functional structures in the head and neck area, the technology bears high therapeutic potential in otorhinolaryngology. OBJECTIVE: In this article, we summarize the concept of CRISPR-Cas9-based therapies, recent achievements in preclinical applications, and future challenges for the implementation of this technology in otolaryngology. MATERIALS AND METHODS: Genetic targeting strategies were analyzed or established using genome sequencing data derived from online databases and literature. RESULTS: Recent research on animal models has shown that genome editing can be used to treat genetic diseases by specifically targeting mutant genomic loci. For example, one preclinical study in the field of otolaryngology has demonstrated that inherited autosomal dominant deafness in mice can be treated using CRISPR-Cas9. Moreover, the same strategies can be used to establish applications for the treatment of head and neck cancer. The greatest challenge appears to be establishment of a system for the safe and efficient delivery of therapeutic nucleotides in clinics. CONCLUSIONS: In theory, genome editing could be used in otolaryngology to target disease-causing genomic loci specifically. However, various challenges have to be overcome until applications can be used clinically.

Long-term outcome of lesional posterior cortical epilepsy surgery in adults.

OBJECTIVE: The aim of this study was to evaluate the short- and long-term seizure outcome and to find predictors of outcome after epilepsy surgery in lesional posterior cortical epilepsies (PCEs). METHODS: The operative outcome in 80 consecutive adult patients with lesional PCEs who underwent resective surgery for intractable partial epilepsy between 1991 and 2006 was retrospectively studied. RESULTS: The probability of remaining in Engel Class I was 66.3% (95% CI 60 to 72) at 6 months, 52.5% (95% CI 47 to 57) at 2 years, 52.9% (CI 45 to 59) at 5 years and 47.1% (CI 42 to 52) at 10 years. Factors predicting poor outcome were the presence of a somatosensory aura, extraregional spikes, incomplete resection, interictal epileptiform discharge (IED) in EEG 6 months and 2 years postsurgery, history of generalised tonic-clonic seizure (GT-CS) and the presence of focal cortical dysplasia in the resected specimen. Factors predicting good outcome were childhood onset of epilepsy, short epilepsy duration, ipsilateral spikes, visual aura, presence of well-circumscribed lesion in preoperative MRI and a pathologically defined tumour. In the multivariate analysis, predictors were different in the long and short term as follows: incomplete resection as proven by postoperative MRI (hazard ratio (HR) 2.059 (CI 1.19 to 3.67)) predicts seizure relapse in short-term follow-up. The presence of IED in the EEG performed 6 months after surgery (HR 2.3 (CI 1.128 to 4.734)) predicts seizure relapse in the long-term fellow-up. However, the absence of a history of GT-CS independently predicts seizure remission in short- and long-term follow-up. CONCLUSIONS: Surgery in PCEs proved to be effective in short- and long-term follow-up. Lesional posterior cortical epilepsy may be a progressive process in a substantial number of cases.

Effects of nifedipine on rhythmic synchronous activity of human neocortical slices.

The antiepileptic effect of the dihydropyridine calcium channel blocker nifedipine was tested in neocortical slice preparations (n=27) from patients ranging in age from four to 46 years (mean=25) who underwent surgery for the treatment of intractable epilepsy. Epileptiform events consisted of spontaneously occurring rhythmic sharp waves as well as of untriggered epileptiform field potentials induced by omission of Mg(2+) from the superfusate, or epileptiform field potentials elicited by application of bicuculline and triggered by single electrical stimuli. (1) Spontaneous rhythmic sharp waves (n=6): with nifedipine (40micromol/l), the repetition rate was decreased down to 30% of initial value, whereas the area under the field potential remained nearly unchanged. (2) Untriggered low Mg(2+) epileptiform field potentials (n=6): with nifedipine (40micromol/l) the area under the field potentials was reduced while the action on the repetition rate was ambiguous. (3) Triggered bicuculline epileptiform field potentials (n=15): with nifedipine (40micromol/l; n=4), no antiepileptic effect was found. There was, however, a marked increase in the area under the epileptiform field potentials. The area under the field potentials was reduced only at a dosage of 60micromol/l (n=11). This effect was stronger when nifedipine was applied with a K(+) concentration raised from 4 to 8mmol/l. The results show that the calcium channel blocker nifedipine is able to reduce differential epileptiform discharges in human neocortical tissue. These observations are in line with previous findings, suggesting that calcium flux into neurons is involved in epileptogenesis. The present results therefore support the idea that some organic calcium antagonists may be useful in human epilepsy therapy, although the etiology of epileptic seizures seems to be a critical factor for the efficacy of the drug.

The effects of verapamil and flunarizine on epileptiform activity induced by bicuculline and low Mg2+ in neocortical tissue of epileptic and primary non-epileptic patients.

In human neocortical slices the specific L-type calcium channel blocker verapamil had been shown to be antiepileptic in the low Mg(2+)-model of epilepsy. The present investigation demonstrated: (1) verapamil exerted also an antiepileptic effect on epileptiform field potentials (EFP) induced by the GABAA-antagonist bicuculline. (2) The unspecific calcium channel modulator flunarizine, which in contrast to verapamil penetrates the blood-brain barrier, depressed EFP in the low Mg(2+)-model and in the bicuculline model. (3) There was no significant difference in the antiepileptic efficacy of verapamil and flunarizine in epileptic (epilepsy surgery) and primary non-epileptic (tumor surgery) neocortical slices.

Flat and steep terminal negativity in the DC-potential after deprivation of oxygen and glucose in human neocortical slices.

The so-called terminal negativity (TN) of the DC-potential is a characteristic reaction of neuronal tissue to hypoxia or ischemia. In a previous study on human neocortical slices, two types of TN with flat and steep slopes of rise (< or >10 mV/min) were found with hypoxia. The aim of the present study was to further investigate causes underlying the occurrence of flat and steep TN. Experiments were performed on 23 human neocortical slices (500 micron) resected from 13 patients (epilepsy and tumour surgery). DC-potential and evoked potentials (white matter stimulation) were recorded in layer III. The extracellular potassium concentration ([K+]o) was measured by K+-sensitive microelectrodes. In an interface type chamber, ischemic episodes were induced by oxygen and glucose deprivation. They were terminated when TN had peaked. Both flat and steep TN also existed with ischemic conditions. There was a linear correlation between the slope of rise of TN and the associated slope of rise in [K+]o, respectively, but none regarding latencies of TN or recovery of evoked potentials. Peak levels in [K+]o were 13.9+/-0.9 mmol/l. Compared to control, the slope of rise and latency of TN were clearly increased by addition of dimethyl sulfoxide (DMSO, 0.4%) to the bath solution, whereas nimodipine (40 micromol/l) in 0.4% DMSO had neither an effect on slope of rise of TN nor on latency of TN. As a whole, our observations suggest, that the actual metabolic state determines the occurrence of flat or steep TN.

Anoxic terminal negative DC-shift in human neocortical slices in vitro.

In animal models, the hallmark of a hypoxic condition is a strong negative shift of the DC potential (anoxic terminal negativity, ATN). This DC-shift is interpreted to be primarily due to a breakdown of the membrane potential of neurons. Such massive neuronal depolarizations have not been reported for all human neocortical neurons in vitro even during prolonged hypoxic periods. This poses the question whether ATN develop also in human neocortical slices made hypoxic. ATN could be observed when human brain slice preparations (n = 15, 13 patients) were subjected to periods of hypoxia (10 to 120 min). These ATN were usually monophasic and appeared with a latency of 16 +/- 4 min (mean +/- S.E.M.). Separating the ATN according to their slopes of rise, steep (> 10 mV/min) and flat (< 10 mV/min) ATN could be distinguished. Steep and flat ATN may be regarded as two different entities of reactions since steep ATN had also greater amplitudes and slopes of decay as compared a flat ATN. With repetitive hypoxias, the latency of both the steep and flat ATN was reduced for the following hypoxic episodes. During hypoxic DC-shifts, evoked potentials were suppressed. With the 1st through 4th hypoxia, they recovered fully within 30 min after reoxygenation when hypoxia was terminated at the plateau of ATN; with extension of hypoxia, recovery was only partial. From the 5th hypoxia onwards, recovery usually did not take place or was not complete.

Spreading depression in human neocortical slices.

Cortical spreading depression (CSD) occurrence has been suggested to be associated with seizures, migraine aura, head injury and brain ischemia-infarction. Only few studies identified CSD in human neocortical slices and no comprehensive study so far evaluated this phenomenon in human. Using the neocortical tissue excised for treatment of intractable epilepsy, we aimed to investigate CSD in human. CSD was induced by KCl injection and by modulating T-type Ca(2+) currents in incubated human neocortical tissues in an interphase mode. The DC-fluctuations were recorded by inserting microelectrodes into different cortical layers. Local injection of KCl triggered single CSD that propagated at 3.1+/-0.1 mm/min. Repetitive CSD also occurred spontaneously during long lasting application (5 h) of the T-type Ca(2+) channel blockers amiloride (50 microM) or NiCl(2) (10 microM) which was concomitant with a reversible extracellular potassium increase up to 50 mM. CSD could be blocked by the N-methyl-D-aspartate receptor antagonist 2-amino-5-phosphonovaleric acid in all cases. The results demonstrate that modulation of the Ca(2+) dynamics conditioned human neocortical slices and increased their susceptibility to generate CSD. Furthermore, these data indicate that glutamatergic pathway plays a role in CSD phenomenon in human.

Vigabatrin reduces epileptiform activity in brain slices from pharmacoresistant epilepsy patients.

Human neocortical temporal lobe tissue resected for treatment of pharmacoresistant epilepsy was investigated. In slices prepared from this tissue, epileptiform field potentials (EFP) were induced by omission of magnesium from the artificial cerebrospinal fluid (ACSF). The effects of the gamma-aminobutyric acid transaminase inhibitor vigabatrin on EFP were tested. Vigabatrin exerted a dose-dependent reduction of the repetition rate of EFP: after 3 h of administration of vigabatrin in concentrations of 100 and 200 micromol/l, the repetition rate of EFP was reduced to 35% and 18% of the initial values, respectively. This effect was not reversible. In control experiments with neocortical slices from rats, vigabatrin reduced EFP in a comparable range. The results demonstrate a strong antiepileptic effect of vigabatrin on EFP in tissues from pharmacoresistant epilepsy patients.

Spatio-temporal distribution of epileptiform activity in slices from human neocortex: recordings with voltage-sensitive dyes.

The spatio-temporal distribution of epileptiform activity was investigated in slices from human temporal neocortex resected during epilepsy surgery. Activity was recorded by use of a voltage-sensitive dye and an optical recording system. Epileptiform activity was induced with 10 microM bicuculline and electrical stimulation of layer I. In 10 slices from six patients investigated, epileptiform activity spread across most of the slice. Largest amplitudes were located in layer II/III. Epileptiform activity was characterized by long-lasting potentials with slow rising phases and a low velocity of spread in the horizontal direction (0.044 m/s). This spatio-temporal pattern of epileptiform activity in human slices was similar to that found previously in neocortical slices from guinea pigs with bicuculline. In four of nine human slices investigated under control bath conditions (in non-epileptogenic medium), the spatio-temporal activity patterns were similar to those of guinea pigs in non-epileptogenic medium. In the remaining five human slices, however, the spread in the horizontal direction was significantly larger (4188 microm) in non-epileptogenic medium than that found in slices from guinea pigs (2171 microm). Activity in human slices showing such 'wide spread' in control bath conditions occasionally had characteristic features of epileptiform activity. Further work will have to clarify whether these epileptiform features reflect intrinsic epileptiform properties in human tissue slices.

Minimally invasive lesionectomies through a stereotactically guided working sleeve.

The authors report on their experience of lesionectomies close to or in the thalamus, basal ganglia, third ventricle and in the temporal lobe. The resection itself is performed stereotactically, MRI or CT guided, either microscopically or endoscopically through a sleeve designed by one of the authors and named PAN working sleeve. Over the last four years this new minimally invasive technique has been successfully applied in 39 cases. Eighteen patients with 11 astrocytoma (6AA, 5All), 5 cavernoma and 2 metastases (melanoma, adenocarcinoma) of the basal ganglion-thalamus area and the trigonum were resected by means of a frontal or an occipital burr-hole, whereby in some cases there were subtotal resections. With four of these patients an existing hemiparesis increased by one degree (according to the proposal of the British Medical Research Council I-V). Seventeen patients with lesions in the foramen Monroi and in the third ventricle also underwent operation by means of frontal access, and in each case there was a total resection. Two of the patients required a shunt due to a persistent hydrocephalus internus. In one of these cases there was intraventricular bleeding which necessitated an intra-operative craniotomy. Four patients with intractable epilepsy were operated through a burr-hole in the anterior area of the os zygomaticum. Three patients were submitted to a selective resection of mesial structures and one to an anterior temporal lobe resection. To date the four patients have had no further seizures and no deficits have been observed.

Evaluation with subdural plates in children and adolescents.

Noninvasive EEG examination is not always adequate for the determination of the epileptogenic area. In such cases invasive methods are required. The authors report on their experience with the implantation of subdural plates for the precise ictal and inter-ictal determination of the epileptogenic areal and the stimulation of the eloquent cortex. From December 1992 to December 1997, 97 patients were evaluated in the Bethel epilepsy center using subdural plates. Of these patients, 44 were children or adolescents, who underwent 45 resections. In order to be able to draw differentiated conclusions on the use of subdural plates in children and adolescents, these patients were divided into three age groups: Group 1, 0-5 years (n = 12); Group 2, 6-11 years (n = 13 + 1 repeat evaluation and resection); Group 3, 12-18 years (n = 19). In the groups of children and adolescents examined there were no complications or progress impediments which might give reason to assume that the application of these techniques involves risks or hazards. This has been verified by the results, in which 75% of age Groups 1 and 3 were categorized as 1 a/b or 2d according to the Engel classification.

Dermoid cyst with crystalline inclusions in adjacent cortex: an ultrastructural study.

Dermoids are rare tumors of the central nervous system. So far, no electron microscopic studies of these tumors have been available. We describe the histology and ultrastructure of a dermoid cyst in a patient with situs inversus and discuss the relationship of keratin and aseptic meningitis, a well-known complication. Histological examination showed an epidermal matrix with 2-4 layers, a cyst containing keratin and debris, some hairs and sebaceous glands. In some areas, chronic inflammation had destroyed the matrix of the cyst wall. Gliosis of the adjacent brain parenchyma was evident, as were eosinophilic rod-shaped crystals. Electron microscopy revealed gliosis with Rosenthal fibers in brain parenchyma adjacent to the tumor capsule. Intracellular osmiophilic, crystalline inclusions were prominent within this area. Glial cells and neuropil were spared. No gross intracellular pathology was seen.

Endoscopic section of the sensory trigeminal root, the glossopharyngeal nerve, and the cranial part of the vagus for intractable facial pain caused by upper jaw carcinoma.

In a patient with intractable facial pain caused by a deep-seated carcinoma of the upper jaw, relief was obtained following endoscopic section of the sensory trigeminal root, the glossopharyngeal nerve, and the cranial part of the vagus. Previous stereotactic thalamotomy had failed. The endoscopic method is briefly described and discussed.

Intercarotid anastomosis with a vein graft in the rat. A model for microsurgical training.

The common carotid arteries were anastomosed through a venous graft in a group of ten rats. The right common carotid artery was ligated below and the left above the anastomosis. In a control group both common carotid arteries (CCA) were ligated. While the animals of the experimental group survived without neurological deficit, those in the control group died 2 hours to 11 days after operation. The anastomoses and brains were studied by light microscopy. The model described is convenient for practising microsurgery, and should constitute part of the routine training program of young neurosurgeons.

Endovascular treatment of trigeminal neuralgia caused by arteriovenous malformation: is surgery really necessary?

A case is presented with secondary trigeminal neuralgia (TN) caused by an arteriovenous malformation (AVM) of the cerebellopontine cistern, which was detected by radiological work-up for planned microvascular decompression. An AVM surrounding the trigeminal nerve was demonstrated on thin-slice heavily T (2)-weighted 3D-sequence on magnetic resonance imaging (MRI) and confirmed by angiography. The first therapeutic step was endovascular embolization with complete obliteration of the AVM and cessation of pain. Nevertheless surgical excision was performed in order to remove compressive vessels and to prevent a recurrence of pain.

Intraparenchymatous ponto-mesencephalic field distribution of brain-stem auditory evoked potentials in man.

Brain-stem auditory evoked potentials (BAEPs) were recorded simultaneously from scalp electrodes and intraparenchymatous sites along a ponto-mesencephalic stereotactic penetration path in a patient with the unique condition of a brain-stem tumour which therapeutically necessitated radionuclide implantation but did not grossly affect scalp BAEPs. The data suggest major contributions to scalp peak III from the pons ipsilateral to the stimulated ear, to peak IV from both ipsi- and contralateral sources, and to peak V mainly from contralateral, more rostral sites. Some local activities evidently did not project to the scalp.

[Respiratory functions in parkinsonian patients with predominant akinesia during treatment with decarboxylase-blocked L-dopa (author's transl)]. / Das Atemverhalten Parkinson-Kranker mit überwiegender Akinese unter der Therapie mit Decarboxylase-geblocktem L-Dopa

The effect of decarboxylase (DC)-blocked L-dopa (proportion of 4: 1) on respiratory function was tested by spirometry in 30 patients with Parkinson's disease. It was possible to improve restrictive ventilatory abnormalities of extra-pulmonary origin (weakness of respiratory musculature). Vital capacity (VC) increased by 0.21 litre. Thisincrease was due to a raised inspiratory capacity and thus an increase in the active part of ventilation. Forced expiration volume (FEV1) was improved by 0.32 litre, FEV1/VC remaining unchanged, which demonstrates that the restrictive ventilatory abnormality was affected. Inspiratory volume (VT) and minute volume at constant respiratory rate increased by 0.07 and 1.171, respectively. These results must be interpreted with caution, but they suggest a raised metabolic level with regained mobility during treatment. All quoted results, except those of respiratory rate and minute volume, were statistically significant (2 mu equals 0.01).

Selective trigeminal root section via an endoscopic transpyramidal retrolabyrinthine approach.

Present experience supports the view that endoscopic access to the cerebellopontine angle has proved an easy and safe therapeutic (selective trigeminal root section in cases of Vth nerve neuralgia) and diagnostic (inspection in cases of otherwise undetectable tumours) method, offering the advantages of little surgical risk and high effectiveness.

Tuberous sclerosis in neurosurgery. An analysis of 18 patients.

Eighteen patients with tuberous sclerosis have been analysed, treated between 1981 and 1987. The diagnosis was made on the clinical triad of adenoma sebaceum, mental retardation and epilepsy. In 6 patients a positive family history of the heredo-familial autosomal-dominant disease was found. The neuroradiological examinations showed in 4 patients tumours with obstruction of the ventricles. All 4 patients were operated on and in 2 additional cases only shunt procedures were performed. The diagnosis, neuroradiological investigations with CT scan and MRI, and the neurosurgical management will be discussed in the light of our material and by literature.

CSF-changes of HVA and 5-HIAA during intermittent and continuous Parkinson therapy with particular regard to prodipin application.

The influence of prodipin, a putative dopamine releasing compound, on the concentration of homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5-HIAA) in the spinal liquor of 28 patients with Parkinson's disease was investigated. The patients were divided into three groups. In group 1 the combined antiparkinson therapy was interrupted, and 20 mg prodipin was infused. In group 2 and 3 the therapy was continued, while an additional 20 mg of prodipin was administered by infusion only to group 3. 4 Liquor-samples were obtained from each patient: 1 basic value and three additional samples 5, 8 and 24 hours after administration of 2 g probenecid. The base concentration of HVA was 15 ng/ml and this was not increased by probenecid in group 1; the concentration of 5-HIAA was 11.6 ng/ml and this was doubled by probenecid to 22.9 ng/ml. The HVA concentration increased to a maximum of 28.9 ng/ml during continued therapy (group 2); the elevated 5-HIAA remained unchanged. Prodipin does not cause an alteration in metabolite concentration in cases of interrupted therapy (group 1), but leads, in the case of continued therapy, to a 1.8-fold increase in HVA, and a 1.6-fold increase in 5-HIAA (group 3).