Next-generation sequencing identifies novel mitochondrial variants in pituitary adenomas.

PURPOSE: Disrupted mitochondrial functions and genetic variants of mitochondrial DNA (mtDNA) have been observed in different human neoplasms. Next-generation sequencing (NGS) can be used to detect even low heteroplasmy-level mtDNA variants. We aimed to investigate the mitochondrial genome in pituitary adenomas by NGS. METHODS: We analysed 11 growth hormone producing and 33 non-functioning [22 gonadotroph and 11 hormone immunonegative] pituitary adenomas using VariantPro™ Mitochondrion Panel on Illumina MiSeq instrument. Revised Cambridge Reference Sequence (rCRS) of the mtDNA was used as reference. Heteroplasmy was determined using a 3% cutoff. RESULTS: 496 variants were identified in pituitary adenomas with overall low level of heteroplasmy (7.22%). On average, 35 variants were detected per sample. Samples harbouring the highest number of variants had the highest Ki-67 indices independently of histological subtypes. We identified eight variants (A11251G, T4216C, T16126C, C15452A, T14798C, A188G, G185A, and T16093C) with different prevalences among different histological groups. T16189C was found in 40% of non-recurrent adenomas, while it was not present in the recurrent ones. T14798C and T4216C were confirmed by Sanger sequencing in all 44 samples. 100% concordance was found between NGS and Sanger method. CONCLUSIONS: NGS is a reliable method for investigating mitochondrial genome and heteroplasmy in pituitary adenomas. Out of the 496 detected variants, 414 have not been previously reported in pituitary adenoma. The high number of mtDNA variants may contribute to adenoma genesis, and some variants (i.e., T16189C) might associate with benign behaviour.

Limitations of high throughput methods for miRNA expression profiles in non-functioning pituitary adenomas.

Microarray, RT-qPCR based arrays and next-generation-sequencing (NGS) are available high-throughput methods for miRNA profiling (miRNome). Analytical and biological performance of these methods were tested in identification of biologically relevant miRNAs in non-functioning pituitary adenomas (NFPA). miRNome of 4 normal pituitary (NP) and 8 NFPA samples was determined by these platforms and expression of 21 individual miRNAs was measured on 30 (20 NFPA and 10 NP) independent samples. Complex bioinformatics was used. 132 and 137 miRNAs were detected by all three platforms in NP and NFPA, respectively, of which 25 were differentially expressed (fold change > 2). The strongest correlation was observed between microarray and TaqMan-array, while the data obtained by NGS were the most discordant despite of various bioinformatics settings. As a technical validation we measured the expression of 21 selected miRNAs by individual RT-qPCR and we were able to validate 35.1%, 76.2% and 71.4% of the miRNAs revealed by SOLiD, TLDA and microarray result, respectively. We performed biological validation using an extended number of samples (20 NFPAs and 8 NPs). Technical and biological validation showed high correlation (p < 0.001; R = 0.96). Pathway and network analysis revealed several common pathways but no pathway showed the same activation score. Using the 25 platform-independent miRNAs developmental pathways were the top functional categories relevant for NFPA genesis. The difference among high-throughput platforms is of great importance and selection of screening method can influence experimental results. Validation by another platform is essential in order to avoid or to minimalize the platform specific errors.

Histopathologic evaluation of aneurysms treated with Guglielmi detachable coils or matrix detachable microcoils.

BACKGROUND AND PURPOSE: The purpose of this study was to evaluate the degree of organization and fibrocellular tissue development in aneurysms treated with bare platinum or biologically active microcoils. METHODS: Twelve aneurysms were removed at autopsy between 1-18 days and another 2 between 2-3 months posttreatment. Four aneurysms were surgically removed between 6 months and 3 years following treatment. One aneurysm removed at 8 days and another at 6 months were treated with bioactive (Matrix) coils; the other 16 with bare platinum (Guglielmi detachable coils; GDCs). All specimens were embedded in plastic, stained with hematoxilin-eosin and elastin and examined by light microscopy. RESULTS: All specimens removed within 3 weeks demonstrated intra-aneurysmal thrombus, without signs of organization or fibrotic tissue formation over the neck regardless of the type of coils used. In the GDC-treated aneurysms, evidence of early thrombus organization was observed within 2-3 months, and completed yet imperfect fibrocellular reaction together with residual thrombus at 2-3 years. In the Matrix-treated specimens, the aneurysm cavity was completely filled with granulation tissue corresponding to still ongoing fibrocellular reaction at 6 months, including newly formed blood vessels, smooth muscle cells, and collagen deposition without signs of residual thrombus. CONCLUSIONS: Our results indicate that in aneurysms treated with bare platinum coils thrombus organization does not occur until late after treatment and may remain imperfect for years. In one aneurysm studied 8 days following treatment with Matrix coils, no difference was noted compared to aneurysms treated with bare platinum coils. In another aneurysm examined 6 months following packing with Matrix coils, the histologic changes support the hypothesis that the biologically active polymer may accelerate aneurysm healing.

Surviving CA1 pyramidal cells receive intact perisomatic inhibitory input in the human epileptic hippocampus.

Temporal lobe epilepsy (TLE) is known to be linked to an impaired balance of excitation and inhibition. Whether inhibition is decreased or preserved in the human epileptic hippocampus, beside the excess excitation, is still a debated question. In the present study, quantitative light and electron microscopy has been performed to analyse the distribution, morphology and input-output connections of parvalbumin (PV)-immunopositive interneurons, together with the entire perisomatic input of pyramidal cells, in the human control and epileptic CA1 region. Based on the degree of cell loss, the patients with therapy-resistant TLE formed four pathological groups. In the non-sclerotic CA1 region of TLE patients, where large numbers of pyramidal cells are preserved, the number of PV-immunopositive cell bodies decreased, whereas axon terminal staining, and the distribution of their postsynaptic targets was not altered. The synaptic coverage of CA1 pyramidal cell axon initial segments (AISs) remained unchanged in the epileptic tissue. The somatic inhibitory input is also preserved; it has been decreased only in the cases with patchy pyramidal cell loss in the CA1 region (control, 0.637; epileptic with mild cell loss, 0.642; epileptic with patchy cell loss, 0.424 microm synaptic length/100 microm soma perimeter). The strongly sclerotic epileptic CA1 region, where pyramidal cells can hardly be seen, contains a very small number of PV-immunopositive elements. Our results suggest that perisomatic inhibitory input is preserved in the epileptic CA1 region as long as pyramidal cells are present. Basket and axo-axonic cells survive in epilepsy if their original targets are present, although many of them lose their PV content or PV immunoreactivity. An efficient perisomatic inhibition is likely to take part in the generation of abnormal synchrony in the non-sclerotic epileptic CA1 region, and thus participate in the maintenance of epileptic seizures driven, for example, by hyperactive afferent input.

Loss of Calbindin-D28K immunoreactivity from dentate granule cells in human temporal lobe epilepsy.

The loss of the calcium binding protein, Calbindin-D28k, from dentate granule cells has been observed in different animal models of epilepsy and in ischaemia. This decrease is accompanied by alterations of calcium and N-methyl-D-aspartate currents, which may explain the hyperexcitability of the dentate gyrus. In the present study, we found a loss of calbindin immunoreactivity from over 90% of the dentate granule cells in lobectomy samples from four of 10 temporal lobe epilepsy patients. In another four patients, over 50%, of dentate granule cells were devoid of calbindin immunoreactivity, whereas the remaining two cases showed a 20-30% decrease. Electron microscopy revealed a normal ultrastructure both in calbindin-containing and calbindin-negative granule cells. Both calbindin-positive and -negative mossy fibre collaterals participated in supragranular sprouting. As inferred from data in animal models, the lack of calbindin in dentate granule cells of human epileptic subjects is likely to result in hyperexcitability of the dentate gyrus, which may then function as a "motor" for seizures.

Changes in the distribution and connectivity of interneurons in the epileptic human dentate gyrus.

The distribution, size, dendritic morphology and synaptic connections of calbindin-, calretinin- and substance P receptor-positive interneurons and pathways have been examined in control and epileptic human dentate gyrus. In the epileptic dentate gyrus, calbindin-containing interneurons are preserved, but their dendrites become elongated and spiny, and several cell bodies appear hypertrophic. The relative laminar distribution of calretinin-containing cells did not change, but their number was considerably reduced. The calretinin-positive axonal bundle at the top of the granule cell layer originating from the supramammillary nucleus expanded, forming a dense network in the entire width of the stratum moleculare. Substance P receptor-immunopositive cells were partially lost in epileptic samples, and in addition, the laminar distribution and dendritic morphology of the surviving cells differed considerably from the controls. In the control human dentate gyrus, the majority of substance P receptor-positive cells can be seen in the hilus, while most are present in the stratum moleculare in the epileptic tissue. Their synaptic input is also changed. The extent of individual pathological abnormalities correlates with each other in most cases. Our data suggest, that although a large proportion of inhibitory interneurons are preserved in the epileptic human dentate gyrus, their distribution, morphology and synaptic connections differ from controls. These functional alterations of inhibitory circuits in the dentate gyrus are likely to be compensatory changes with a role to balance the enhanced excitatory input in the region.

Synaptic reorganization of calbindin-positive neurons in the human hippocampal CA1 region in temporal lobe epilepsy.

The distribution, morphology, synaptic coverage and postsynaptic targets of calbindin-containing interneurons and afferent pathways have been analyzed in the control and epileptic CA1 region of the human hippocampus. Numerous calbindin-positive interneurons are preserved even in the strongly sclerotic CA1 region. The morphology of individual cells is altered: the cell body and dendrites become spiny, the radially oriented dendrites disappear, and are replaced by a large number of curved, distorted dendrites. Even in the non-sclerotic epileptic samples, where pyramidal cells are present and calbindin-immunoreactive interneurons seem to be unchanged, some modifications could be observed at the electron microscopic level: they received more inhibitory synaptic input, and the calbindin-positive excitatory afferents - presumably derived from the CA1, the CA2 and/or the dentate gyrus - are sprouted. In the strongly sclerotic tissue, with the death of pyramidal cells, calbindin-positive terminals (belonging to interneurons and the remaining excitatory afferents) change their targets. Our data suggest that an intense synaptic reorganization takes place in the epileptic CA1 region, even in the non-sclerotic tissue, before the death of considerable numbers of pyramidal cells. Calbindin-positive interneurons participate in this reorganization: they show plastic changes in response to epilepsy. The enhanced inhibition of inhibitory interneurons may result in the disinhibition of pyramidal cells or in an abnormal synchrony in the output region of the hippocampus.

GABAergic interneurons are the targets of cannabinoid actions in the human hippocampus.

Cannabinoids have been shown to disrupt memory processes in mammals including humans. Although the CB1 neuronal cannabinoid receptor was identified several years ago, neuronal network mechanisms mediating cannabinoid effects are still controversial in animals, and even more obscure in humans. In the present study, the localization of CB1 receptors was investigated at the cellular and subcellular levels in the human hippocampus, using control post mortem and epileptic lobectomy tissue. The latter tissue was also used for [3H]GABA release experiments, testing the predictions of the anatomical data. Detectable expression of CB1 was confined to interneurons, most of which were found to be cholecystokinin-containing basket cells. CB1-positive cell bodies showed immunostaining in their perinuclear cytoplasm, but not in their somadendritic plasmamembrane. CB1-immunoreactive axon terminals densely covered the entire hippocampus, forming symmetrical synapses characteristic of GABAergic boutons. Human temporal lobectomy samples were used in the release experiments, as they were similar to the controls regarding cellular and subcellular distribution of CB1 receptors. We found that the CB1 receptor agonist, WIN 55,212-2, strongly reduced [3H]GABA release, and this effect was fully prevented by the specific CB1 receptor antagonist SR 141716A. This unique expression pattern and the presynaptic modulation of GABA release suggests a conserved role for CB1 receptors in controlling inhibitory networks of the hippocampus that are responsible for the generation and maintenance of fast and slow oscillatory patterns. Therefore, a likely mechanism by which cannabinoids may impair memory and associational processes is an alteration of the fine-tuning of synchronized, rhythmic population events.

Surgical experience with frontolateral keyhole craniotomy through a superciliary skin incision.

OBJECTIVE: The purpose of this study was to evaluate the results of 173 frontolateral keyhole minicraniotomies performed on 155 patients with aneurysms of the anterior or posterior cerebral circulation and for supratentorial tumors. METHODS: The frontolateral keyhole craniotomy is a modification of the generally used pterional approach. Of the 155 patients studied, 102 harbored saccular arterial aneurysms in the vessels of the anterior or posterior cerebral circulation, and 53 had various tumors in the frontal base, suprasellar, or parasellar region. The operations were carried out through an approximately 2.5- x 3-cm frontolateral miniaturized craniotomy after a skin incision just above the eyebrow. RESULTS: Despite the small size of the craniotomy, the exploration allows enough room for intracranial manipulation with maximal protection of the brain and other intracranial structures. The presented series of patients did not have any craniotomy-related complications. CONCLUSION: In our experience, the frontolateral keyhole craniotomy, together with the advent of the modern neuroanesthesia, cerebrospinal fluid drainage, and microsurgical techniques, is a safe approach for an experienced neurosurgeon to use in the treatment of supratentorial aneurysms or tumors of the anterior fossa and sellar regions.

Primary meningeal melanocytoma of the pineal region. Case report.

A unique case of a meningeal melanocytoma located in the pineal region is presented. This 48-year-old man presented with a round pineal region tumor that caused triventricular hydrocephalus and exhibited slow clinical progression. During surgery a black encapsulated tumor was found and totally removed. On histopathological examination, the tumor proved to be a meningeal melanocytoma. In this report cell culture data are presented and the relevant literature is reviewed. The problems of histopathological diagnosis and management of patients with melanocytomas are also discussed.

Third ventricle germinoma after total removal of intrasellar teratoma. Case report.

A unique case is presented of a third ventricle germinoma developing 3 years after total removal of an intrasellar teratoma. The third ventricle germinoma was not considered to be a recurrence or dissemination of the mature intrasellar teratoma but to have been transformed from multicenter germ cells present in the midline of the brain with different temporal development. The relevant literature is reviewed and the problems of management of patients with germ-cell tumors are discussed.

Apoplexy of a pituitary macroadenoma as a severe complication of preoperative thyrotropin-releasing hormone (TRH) testing.

The case history of a 54-year-old male suffering from pituitary macroadenoma with suprasellar extension is reported. A TRH-test with 200 micrograms i.v. was followed by severe headache and vomiting after 60', and by development of ophthalmoplegia on the following day. Hyperdens patches on the CT scan showed haemorrhage into the tumor. A chromophobic adenoma with macroscopic and histological signs of haemorrhage was removed via the transsphenoidal route. In the postoperative period the ophthalmoplegia gradually disappeared but central hypoadrenia and hypothyroidism occurred. This is the second case in the literature showing that TRH alone and in a low dose may cause pituitary tumor apoplexy. It is concluded that TRH-testing is a risk for the patient with pituitary apoplexy. If, due to the size of the tumor the patients have to be operated on in any case, and the test is not of essential diagnostic value, the TRH-test should be done only in selected cases. Its use in the postoperative evaluation however is without risk for the patients.

Management of pineal region tumours.

Pineal region tumours represent a colourful, challenging peculiarity of brain pathology. Views on their management are still much divided and controversial. Data of fifty patients with the whole palette of these tumours seen in the National Institute of Neurosurgery have been analysed in view of the result of management versus histology of these tumours. Findings of tumour marker studies have not at all been conclusive in predicting histology and outcome, however, cytology of the cerebrospinal fluid (CSF), if positive, pointed toward a very gloomy management result in all cases. Merits of infratentorial-supracerebellar, occipito-transtentorial approaches of direct surgery, palliative interventions and their timing, as well as that of irradiation are discussed in comparison with opinions and arguments from the literature. Shunt procedures alone proved to be dangerous in some cases by evoking haemorrhagic complications. In carefully selected cases microsurgical intervention gave the best possible results in expansively growing pineal region tumours. There is still place for irradiation and chemotherapy, again, in certain types of mass lesions.

Experiences in the endovascular treatment of cerebral arteriovenous angiomas.

Percutaneous superselective balloon catheterization for the treatment of large cerebral arteriovenous malformations, for which direct operation is infeasible, is described. Both a detachable balloon technique and a "calibrated leak" free embolization have been applied, based on a new hardening silicone material elaborated by the authors. The experiences of operations performed since 1978 are represented by four selected case studies.

Transformation of a human mixed GH-PRL adenoma cell population in response to long-term bromocriptine treatment.

A child was operated 3 times because of a recurrent growth hormone- and prolactin-producing pituitary adenoma. Between the operations she was treated for five years with bromocriptine. The characteristics of the tumour cell population collected after the last operation was now examined by electron microscopy, immunocytochemistry, and in tissue culture and compared to those of the primary tumour cells reported earlier. A prominent change was the reversal of the proportion of the densely and sparsely granulated cells in favour of the GH-type, densely granulated cells. These cells, some of them coexpressing PRL, did not essentially change their characteristics either in vivo or in culture. On the other hand, pleomorphous cells with smaller cytoplasmic area and prominent lysosomal structures represented the sparsely granulated population containing PRL or both PRL and GH. The morphological alteration of the PRL-type cells was also reflected in vitro. Hence, while GH-type cells prevail, at least a sub-population of PRL-type cells survives long-term bromocriptine administration. A shift in the incidence of the two cell types in favour of the GH-type cells explains the change in the endocrine status of the patient.

Effect of vasoactive intestinal polypeptide (VIP) on growth hormone (GH) and prolactin (PRL) release and cell morphology in human pituitary adenoma cell cultures.

Six GH adenomas and three prolactinomas were investigated by light- and electron-microscopic morphological and immunocytochemical methods and the effect of vasoactive intestinal polypeptide (VIP) on growth hormone (GH) and prolactin (PRL) secretion was tested in vitro. The tumour cells of the acromegalic patients revealed both GH and PRL immunoreactivity while prolactinomas showed only PRL activity. All the adenomas stained immunocytochemically also for VIP. By electron microscopy, the tumours included two densely and two sparsely granulated GH, two mixed GH/PRL, and three sparsely granulated PRL adenomas. The dissociated cells were explanted, and cultured in vitro. The cultures in micro test plates were treated with VIP at different concentrations between 10(-5)-10(-12) M. GH and PRL contents in the culture media were measured by radioimmunoassay. GH release was significantly stimulated by VIP in a dose-dependent manner over the whole concentration range, while VIP was effective on the PRL release only at 10(-6)-10(-7) M concentration. The cells of a mixed adenoma were grown in Petri dishes and used for ultrastructural and immunocytochemical studies. The cytoplasmic structure of the cells treated with VIP corresponded to that of active hormone-secreting cells with large ergastoplasmic fields and Golgi zones containing secretory granules. Massive exocytotic events were encountered mainly in the GH-type cells. GH and PRL double immunocytochemistry showed the predominance of GH cells, many of them containing low amounts of PRL as well. Cells predominantly containing PRL were spread among them, they also might contain GH as well. Some of the cells contained only a single immunoreactive hormone. The intensity of gold labelling of the secretory granules appeared higher in the VIP-treated cells than in the untreated control ones which showed a cytoplasmic structure characteristic of glandular cells with low secretory activity. As all the adenoma cells both contained and reacted to VIP, our results are in agreement with an autocrine or paracrine effect of this peptide. The fine structure of the cells in the cultures treated with VIP supply an additional argument to the assumption that VIP may serve as a growth factor for these cell types.

Early surgery for ruptured cerebral aneurysm.

Preliminary experience with 150 consecutive cases of ruptured cerebral aneurysms operated on within 48 hours is reported. The rationale of this emergency procedure is to prevent early rerupture and also to prevent neurological ischaemic consequences of the subarachnoid haemorrhage likely to develop in the first week after a rupture. The acceptable outcome of the surgically treated cases (87% excellent and good outcome) has been favourably matched to those of a group of equal number of consecutive cases seen in suitable condition for surgery within 48 hours by medical personnel but that remained unoperated for various reasons. The incidence of delayed neurological ischaemia as the definite cause of death is lower in the group operated on in the acute stage than those with delayed surgery, although the overall incidence of clinical vasospasm does not seem significantly lower than in the delayed surgery group.

[Persistent trigeminal artery associated with pituitary adenoma]. / Hypophysis adenomával társult persistáló intrasellaris arteria trigemini primitiva.

The authors report of a case with a pituitary macroadenoma which was associated with a persistent trigeminal artery. The tumour was removed by transsphenoidal microsurgical approach to the sella turcica. To recognise the developmental anomaly is very important to avoid complications during operation.

[A new technic in the management of hydrocephalus: neuro-endoscopy]. / Uj esély az agyvíz keringési zavarok kezelésében: neuroendoszkópia.

Conventional valve shunting for treatment of hydrocephalus has a high rate of long-term complication. After a brief historical review of neurosurgical endoscopy the authors present the different indications and methods of neuroendoscopy. Between 1995 and 1997 twenty-two pediatric patients underwent endoscopic surgery at National Institute of Neurosurgery. Twelve of them had third ventriculo-cistemostomy, and cystic wall fenestration was performed in ten children. All but one patient benefited from this minimally invasive endoscopic technic. Minor transient complications were seen in three cases, and only one patient had long-term pupillary dilatation due to the surgical procedure.

[Glycoprotein hormone alpha-subunit secretion in non-functioning pituitary adenomas]. / Glikoprotein hormon alpha-alegység elválasztás nem funkcionáló hypophysisdaganatokban.

The aim of the present study was to investigate the prevalence of elevated free glycoprotein hormone alpha-subunit in different pituitary adenomas, to establish the diagnostic value of the basal and stimulated free alpha-subunit secretion in non-functioning adenomas. Serum basal levels of alpha-subunit were increased in 1 of 22 untreated, in 1 of 16 operated patients with non-functioning adenoma, in 6 of 28 untreated, in 1 of 7 operated patients with acromegaly, in 0 of 5 untreated prolactinomas and in 0 of 1 untreated gonadotrop adenoma. Overall free alpha-subunit levels were increased in 9 of 79 cases (11.4%). In 6 of 9 patients with untreated non-functioning adenoma thyrotrop hormone releasing hormone caused an abnormal--paradox--elevation of serum alpha-subunit. These data indicate that measurement of basal and stimulated alpha-subunit is of relatively poor value in the diagnosis of non-functioning pituitary adenomas. The transsphenoidal surgery did not resulted in a change of alpha-subunit secretion neither in patients with non-functioning adenoma nor with acromegaly. The present data confirm the view that non-functioning pituitary adenomas are not homogeneous since this subset of tumors includes adenomas that either do not secrete measurable amounts of free alpha-subunit or produce normal or supranormal amounts of subunits as consequence of still undefined biosynthetic abnormalities.