In vivo assessment of the human cerebral microcirculation and its glycocalyx: A technical report.

INTRODUCTION: The cerebral microcirculation and its glycocalyx, a matrix coating the luminal endothelium, are key regulators of capillary permeability and cerebral blood flow. Microvascular abnormalities are described in several neurological disorders. However, assessment of the cerebral microcirculation and glycocalyx has mainly been performed ex vivo. NEW METHOD: Here, the technical feasibility of in vivo assessment of the human cerebral microcirculation and its glycocalyx using sidestream dark field (SDF) imaging is discussed. Intraoperative assessment requires the application of a sterile drape covering the camera (slipcover). First, sublingual measurements with and without slipcover were performed in a healthy control to assess the impact of this slipcover. Subsequently, using SDF imaging, the sublingual (reference), cortical, and hippocampal microcirculation and glycocalyx were evaluated in patients who underwent resective brain surgery as treatment for drug-resistant temporal lobe epilepsy. Finally, vessel density, and the perfused boundary region (PBR), a validated gauge of glycocalyx health, were calculated using GlycoCheck© software. RESULTS: The addition of a slipcover affects vessel density and PBR values in a control subject. The cerebral measurements in five patients were more difficult to obtain than the sublingual ones. This was probably at least partly due to the introduction of a sterile slipcover. Results on vessel density and PBR showed similar patterns at all three measurement sites. COMPARISON WITH EXISTING METHODS: This is the first report on in vivo assessment of the human cerebrovascular glycocalyx. Assessment of the glycocalyx is an additional application of in vivo imaging of the cerebral microcirculation using SDF technique. This method enables functional analysis of the microcirculation and glycocalyx, however the addition of a sterile slipcover affects the measurements. CONCLUSIONS: SDF imaging is a safe, quick, and straightforward technique to evaluate the functional cerebral microcirculation and glycocalyx. Because of their eminent role in cerebral homeostasis, this method may significantly add to research on the role of vascular pathophysiology underling various neurological disorders.

Validation of reference genes in human chordoma.

BACKGROUND: Chordoma are rare slow-growing tumors of the axial skeleton, which are thought to arise from remnants of the notochord. Little is known about the underlying mechanisms that drive this tumor. However, the assessment of gene expression levels by quantitative real-time polymerase chain reaction (qRT-PCR) is hampered due to a lack of validated reference genes. Using an unstable reference gene in qRT-PCR may lead to irreproducible results. METHODS: The expression of 12 candidate reference genes (ACTB, B2M, T, EF1a, GAPDH, HPRT, KRT8, KRT19, PGK1, RS27a, TBP, and YWHAZ) was analyzed by qRT-PCR in flash frozen chordoma samples from 18 patients. GeNorm and NormFinder algorithms were used to rank the stability of the genes. RESULTS: From most to least stably expressed, the top six genes found by geNorm were PGK1, YWHAZ, ACTB, HPRT, EF1A, and TBP. When analyzed by NormFinder, the top six genes were ACTB, YWHAZ, PGK1, B2M, TBP, and HPRT. GAPDH alone, which is often used as a reference gene in chordoma gene expression studies, is not stable enough for reliable results. CONCLUSION: In gene expression studies of human chordomas, PGK1, ACTB, and YWHAZ are more stably expressed, and therefore, are preferred reference genes over the most often used reference gene so far, GAPDH.

Protocol for intraoperative assessment of the human cerebrovascular glycocalyx.

INTRODUCTION: Adequate functioning of the blood-brain barrier (BBB) is important for brain homoeostasis and normal neuronal function. Disruption of the BBB has been described in several neurological diseases. Recent reports suggest that an increased permeability of the BBB also contributes to increased seizure susceptibility in patients with epilepsy. The endothelial glycocalyx is coating the luminal side of the endothelium and can be considered as the first barrier of the BBB. We hypothesise that an altered glycocalyx thickness plays a role in the aetiology of temporal lobe epilepsy (TLE), the most common type of epilepsy. Here, we propose a protocol that allows intraoperative assessment of the cerebrovascular glycocalyx thickness in patients with TLE and assess whether its thickness is decreased in patients with TLE when compared with controls. METHODS AND ANALYSIS: This protocol is designed as a prospective observational case-control study in patients who undergo resective brain surgery as treatment for TLE. Control subjects are patients without a history of epileptic seizures, who undergo a craniotomy or burr hole surgery for other indications. Intraoperative glycocalyx thickness measurements of sublingual, cortical and hippocampal microcirculation are performed by video microscopy using sidestream dark-field imaging. Demographic details, seizure characteristics, epilepsy risk factors, intraoperative haemodynamic parameters and histopathological evaluation are additionally recorded. ETHICS AND DISSEMINATION: This protocol has been ethically approved by the local medical ethical committee (ID: NL51594.068.14) and complies with the Declaration of Helsinki and principles of Good Clinical Practice. Informed consent is obtained before study enrolment and only coded data will be stored in a secured database, enabling an audit trail. Results will be submitted to international peer-reviewed journals and presented at international conferences. TRIAL REGISTRATION NUMBER: NTR5568.

Notochord isolation using laser capture microdissection.

BACKGROUND: Chordoma are malignant tumors of the axial skeleton, which arise from remnants of the notochord. The Notochord (chorda dorsalis) is an essential embryonic structure involved in the development of the nervous system and axial skeleton. Therefore, the notochord seems to be the most biologically relevant control tissue to study chordoma in molecular biology research. Nevertheless, up to now mainly different tissues but not the notochord have been used as control for chordoma, due to difficulty of isolating notochordal tissue. Here, we describe a fast and precise method of isolating notochordal cells. METHODS: Examination of human fetuses, with a gestation of 9, 11 and 13 weeks, using (immuno)histochemical methods was performed. To isolate pure notochord cells for further molecular biology investigation five flash frozen fetuses between 9 and 10 weeks of gestation were dissected by microtome slicing. Thereafter pure notochord cells for further molecular biology investigation where harvested by using laser capture microdissection (LCM). RNA was extracted from these samples and used in quantitative PCR. RESULTS: This study illustrates notochord of embryonic spines in three different stages of gestation (9-11-13 weeks). Immunohistochemical staining with brachyury showed strong staining of the notochord, but also weak staining of the intervertebral disc and vertebral body. LCM of notochord slices and subsequent total RNA extraction resulted in a good yield of total RNA. qPCR analysis of two housekeeping genes confirmed the quality of the RNA. CONCLUSION: LCM is a fast and precise method to isolate notochord and the quality and yield RNA extracted from this tissue is sufficient for qPCR analysis. Therefore early embryo notochord isolated by LCM is suggested to be the gold standard for future research in chordoma development, classification and diagnosis.

A vertebral extra dural chordoma at C5, possibly deriving from a clival chordoma.

BACKGROUND: Clival chordomas are a rare type of cancer with low metastatic potential and primary metastasize to the lung or bones. CASE DESCRIPTION: This case report describes a possible metastatic, paravertebral chordoma at level C4-C5 in a patient with a past medical history of a clival chordoma. CONCLUSION: Chordomas are unpredictable and may metastasise.

Deep brain stimulation in tinnitus: current and future perspectives.

Chronic tinnitus, also known as ringing in the ears, affects up to 15% of the adults and causes a serious socio-economic burden. At present, there is no treatment available which substantially reduces the perception of this phantom sound. In the past few years, preclinical and clinical studies have unraveled central mechanisms involved in the pathophysiology of tinnitus, replacing the classical periphery-based hypothesis. In subcortical auditory and non-auditory regions, increased spontaneous activity, neuronal bursting and synchrony were found. When reaching the auditory cortex, these neuronal alterations become perceptually relevant and consequently are perceived as phantom sound. A therapy with a potential to counteract deeply located pathological activity is deep brain stimulation, which has already been demonstrated to be effective in neurological diseases such as Parkinson's disease. In this review, several brain targets are discussed as possible targets for deep brain stimulation in tinnitus. The potential applicability of this treatment in tinnitus is discussed with examples from the preclinical field and clinical case studies.

Implementation of a mobile 0.15-T intraoperative MR system in pediatric neuro-oncological surgery: feasibility and correlation with early postoperative high-field strength MRI.

INTRODUCTION: We analyze our preliminary experience using the PoleStar N20 mobile intraoperative MR (iMR) system as an adjunct for pediatric brain tumor resection. METHODS: We analyzed 11 resections in nine children between 1 month and 17 years old. After resection, we acquired iMR scans to detect residual tumor and update neuronavigation. We compared final iMR interpretation by the neurosurgeon with early postoperative MR interpretation by a neuroradiologist. RESULTS: Patient positioning was straightforward, and image quality (T1 7-min 4-mm sequences) sufficient in all cases. In five cases, contrast enhancement suspect for residual tumor was noted on initial postresection iMR images. In one case, a slight discrepancy with postoperative imaging after 3 months was no longer visible after 1 year. No serious perioperative adverse events related to the PoleStar N20 were encountered, except for transient shoulder pain in two. CONCLUSIONS: Using the PoleStar N20 iMR system is technically feasible and safe for both supra- and infratentorial tumor resections in children of all ages. Their small head and shoulders favor positioning in the magnet bore and allow the field of view to cover more than the area of primary interest, e.g., the ventricles in an infratentorial case. Standard surgical equipment may be used without significant limitations. In this series, the use of iMR leads to an increased extent of tumor resection in 45 % of cases. Correlation between iMR and early postoperative MR is excellent, provided image quality is optimal and interpretation is carefully done by someone sufficiently familiar with the system.

Gamma knife radiosurgery for vestibular schwannomas: identification of predictors for continued tumor growth and the influence of documented tumor growth preceding radiation treatment.

OBJECTIVES/HYPOTHESIS: Gamma knife radiosurgery (GKRS) has become an important treatment modality for vestibular schwannomas. The primary aim of this study was to investigate whether tumor growth at the moment of GKRS has any correlation with the outcome. The secondary aim was to identify clinical predictors of radioresistance in vestibular schwannoma patients treated with GKRS. STUDY DESIGN: One hundred vestibular schwannoma patients, treated with GKRS, were divided into two groups: 1) proven tumor growth preceding GKRS; and 2) previous history of growth unknown. GKRS outcome was defined in two ways. According to the first definition, GKRS was said to have failed when additional treatment had taken place. According to the second one, a volume decrease >20% after 2 years marked successful treatment. METHODS: Correlations between outcome and growth status were determined with SPSS software. Furthermore, the study assessed how different variables (patient data, history, tumor characteristics, imaging, and audiovestibular examinations) correlated with the outcome of GKRS. RESULTS: No significant difference regarding success and failure of GKRS was found between the two patient groups. The mean reduction in tumor volume after GKRS was less pronounced in patients in whom tumor growth was demonstrated before treatment, but this finding was not significant. No significant predictors (P < .05) could be identified in this data set. CONCLUSIONS: This study found no indication that growth at the moment of GKRS influences therapeutic outcome, nor did it identify any predictors of the outcome after GKRS in vestibular schwannoma patients.

The impact of auditory feedback on neuronavigation.

OBJECT: We aimed to develop an auditory feedback system to be used in addition to regular neuronavigation, in an attempt to improve the usefulness of the information offered by neuronavigation systems. INSTRUMENTATION: Using a serial connection, instrument co-ordinates determined by a commercially available neuronavigation system were transferred to a laptop computer. Based on preoperative segmentation of the images, the software on the laptop computer produced an audible signal whenever the instrument moved into an area the surgeon wanted to avoid. METHODS: To evaluate the impact of our setup on volumetric resections, phantom experiments were conducted. CT scans were acquired from eight blocks of floral foam. In each of these scans, a target-volume was segmented. This target-volume was subsequently resected using either regular neuronavigation or neuronavigation extended with auditory feedback. A 'postoperative' CT scan was used to compare the resection cavity to the preoperatively planned target-volume. FINDINGS: The resemblance between the resection cavity and the target-volume was greater each time auditory feedback had been used. This corresponded with more complete removal of the target-volume. However, it also corresponded with the removal of more non-target 'tissue' in two out of four cases. CONCLUSIONS: The usefulness of auditory feedback was made plausible and the use of a new type of navigation phantom was illustrated. Based on these results, we recommend incorporation of auditory feedback in commercially available neuronavigation systems, especially since this is relatively inexpensive.

Subtyping of oligo-astrocytic tumours by comparative genomic hybridization.

Oligo-astrocytic tumours (OAs) histologically show both oligodendroglial and astrocytic differentiation. Unequivocal criteria for delineation of OAs from pure oligodendroglial (Os) and astrocytic (As) tumours and for grading of OAs are lacking. Molecular genetic analysis may allow for a better characterization of OAs and thereby guide prognostic and therapeutic decisions. Comparative genomic hybridization (CGH) was performed on 39 gliomas with variable phenotypic expression of histological features characteristic of both astrocytic and oligodendroglial differentiation. The results show that OAs are genetically more heterogeneous than Os. In addition to the "-1p/-19q" and "+7/-10" subtypes that have been previously recognized, two additional genetic subtypes, "intermediate" and "other", were identified in the present study. "Intermediate" OAs likely represent progression from "-1p/-19q" tumours. The "other" subtype appears to represent an additional, heretofore unrecognized, genetic pathway(s). Application of rigorously "strict" histopathological criteria, as opposed to "relaxed" criteria, for the selection of oligo-astrocytic tumours resulted in a higher percentage of "-1p/-19q" tumours, but some "-1p/-19q" tumours might be missed. The results suggest that molecular genetic analysis is a useful and valid additional tool for the classification of gliomas, particularly for the significant subset of tumours in which subjective histopathological criteria are insufficient for an unequivocal distinction between Os, As, and mixed OAs.

PTEN mutation analysis in two genetic subtypes of high-grade oligodendroglial tumors. PTEN is only occasionally mutated in one of the two genetic subtypes.

We recently identified two genetic subtypes of high-grade oligodendroglial tumors (HG-OT): 1p-/19q- HG-OT are characterized by a loss of chromosome 1p32-36 (del(1)(p32-p36) and/or a del(19)(q13. 3); whereas +7/-10 HG-OT harbor a gain of chromosome 7 (+7) and/or a -10 without a loss of 1p32-36 and 19q13.3. Because a -10 and a +7 are most frequently detected in glioblastomas (GBM), the genotype of +7/-10 HG-OT suggests that these tumors are GBM with a prominent oligodendroglial phenotype rather than anaplastic oligodendrogliomas. PTEN is a tumor suppressor gene, located at 10q23.3, which is involved in tumor progression of GBM and other neoplasms. In this study, we screened for PTEN mutations in six low-grade oligodendroglial tumors (LG-OT), five 1p-/19q- HG-OT, seven +7/-10 HG-OT, and nine xenografted GBM. PTEN mutations were detected in none of the LG-OT and 1p-/19q- HG-OT, once in +7/-10 HG-OT, and frequently in GBM. As one of the +7/-10 HG-OT harbored a PTEN mutation, this demonstrates that PTEN can be involved in the oncogenesis of this genetic subtype of HG-OT. The lower frequency of PTEN mutations in +7/-10 HG-OT compared to GBM suggests that these tumors are of a distinct tumor type rather than GBM. Published by Elsevier Science Inc.

Genetic reflection of glioblastoma biopsy material in xenografts: characterization of 11 glioblastoma xenograft lines by comparative genomic hybridization.

OBJECT: Human tumors implanted as subcutaneous xenografts in nude mice are widely used for the study of tumor biology and therapy. Validation of these models requires knowledge of the genetic makeup of the xenografts. The aim of this study was to establish whether chromosomal imbalances in 11 xenograft lines derived from human glioblastomas multiforme (x-GBMs) are similar to those found in GBM biopsy samples. The authors also studied genetic stability during serial passaging of three xenograft lines. METHODS: Chromosomal imbalances in x-GBMs were detected using comparative genomic hybridization (CGH). The authors compared the CGH results in x-GBMs with those in the original GBMs (o-GBMs) that were used to establish three of the xenograft lines and with the GBM biopsy results reported in the literature (1-GBMs). In three xenograft lines two different passages were analyzed. CONCLUSIONS: The results show that the chromosomal imbalances in x-GBMs are similar to those in o-GBMs and 1-GBMs, indicating that the GBM xenograft lines used were valid models from a genetic point of view. The CGH analysis of two different passages of three xenograft lines indicates that x-GBMs (like 1-GBMs) show intratumoral genetic heterogeneity and do not acquire chromosomal imbalances as a result of serial passaging.

[Cavernous sinus syndrome]. / Het sinus-cavernosussyndroom.

A clear and concise description and clinical interpretation of the cavernous sinus syndrome are lacking. Pathological changes in or around the cavernous sinus may lead to failure of eye muscle nerves and of one or more branches of the trigeminal nerve. The clinical signs of the cavernous sinus syndrome are combinations of failure of these cranial nerves sometimes with exophthalmus. Because many nerves can be wholly or partially involved in the syndrome, there is no clinical uniformity and the cavernous sinus syndrome has never been well defined. A neurotopographical classification is proposed in order to simplify the multiple interpretations of the cavernous sinus syndrome. The classical cavernous sinus syndrome is divided into three syndromes: the syndrome of the superior orbital fissure, the syndrome of the lateral wall of the cavernous sinus and the central cavernous sinus syndrome.

Surgical approach and histoanatomical aspects of the oculomotor nerve in rats.

The surgical approach and some histoanatomical characteristics of the intracavernous portion of the oculomotor nerve are described. Moreover, some perioperative precautions for intracranial surgical procedures in the rat are reported and the suitability of the rat as a model for studying intracranial nerve regeneration is discussed. With the data provided, this model of oculomotor nerve approach can be used to study various aspects of intracranial nerve regeneration.

End-to-end versus peripheral nerve graft repair of the oculomotor nerve in rats: A comparative histological and morphometric study.

A comparative study was undertaken to evaluate end-to-end versus peripheral nerve graft repair in cranial nerve reconstruction. In 14 rats, the oculomotor nerve was sharply transected in the cavernous sinus and repaired either by end-to-end coaptation (n = 7) or by interposition of a peripheral nerve graft (n = 7). The results were evaluated 16 weeks after surgery by light and transmission electron microsurgery and by morphometric analysis. The degree of neuroma formation, fibrosis, and axonal disorganisation at the repair site was the same for both groups. Histologically, both end-to-end and graft repair groups revealed various degrees of axonal regeneration with myelinated nerve fibres in the distal nerve segments. In both groups, the number of nerve fibres distal to the repair site was increased compared to proximal to the repair (P < 0.001) but myelinated axon diameter was significantly less than that of control nerves (P < 0.001). No difference existed between the two repair groups in terms of mean myelinated axonal diameter. However, the number and density of myelinated axons was statistically greater in the graft group (P < 0.05). In conclusion, despite the disadvantage of two repair sites, peripheral nerve grafting results in equal or slightly superior axonal regeneration compared to an end-to-end repair in the rodent model of intracranial oculomotor nerve reconstruction. We speculate that this may be due to the structure of the peripheral nerve graft.

Effect of a modified Nd:YAG laser technique on neuroma formation: An experimental study in rat sciatic nerve.

BACKGROUND AND OBJECTIVES: Traumatic transection of a peripheral nerve is inherently associated with the development of neuroma at the end of the proximal stump, often leading to therapy-resistant pain. This study was designed to evaluate whether the neodymium:yttrium aluminum garnet (Nd:YAG) laser could prevent neuroma formation after neurectomy. STUDY DESIGN/MATERIALS AND METHODS: The sciatic nerves of 14 rats were diffuse coagulated by defocused Nd:YAG laser (12 W power), and subsequently transected with additional focused laser energy. The control group consisted of contralateral nerves transected by microscissors. The nerves were reexposed at different time intervals up to 9 weeks after surgery, and evaluation consisted of macroscopy, and light and transmission electron microscopy. RESULTS: True neuroma formation could not be observed after laser transection, and only five nerves formed a neuromatous bulb, with minimal adhesions to surrounding tissue. Microscissor transection resulted in widespread amputation neuromas, consisting of regenerating axons and connective tissue, and nervous tissue regenerating into surrounding tissue. Laser-transected nerves showed degenerative changes of the axons and myelin, while proliferation of Schwann cells could not be observed. No outgrowth of axons could be observed outside the coagulated proximal stump. An epi/perineurial layer was present, covering the nerve stumps. Microscissor-transected nerves showed proliferation of fibroblasts and Schwann cells, forming minifascicles, and vigorous outgrowth of axons into the tissue and even into the distal nerve stump. CONCLUSIONS: Within the limitations of this study it is concluded that the formation of amputation neuromas is suppressed by Nd:YAG laser application by thermal coagulation of the nerve and suppression of Schwann-cell proliferation.

A new technique for the assessment of the draining area of a cerebral vein.

BACKGROUND: At present, it is not exactly clear which vein is allocated for drainage of blood to a particular area of the human brain. Knowledge of these draining areas is very important for the understanding of occlusive venous diseases. A method was developed that offers the possibility to investigate the draining area of a cerebral vein, with the help of an animal model. METHODS: Brains of sacrificed rabbits are removed and are anterogradely perfused with a coloring matter. Then a vein chosen at random is occluded and anterograde perfusion is restarted using another coloring substance. The working hypothesis is that the part of the brain that is solely dependent for its drainage of blood upon the occluded vein (the draining area of the vein) will show a deficit in staining after the second perfusion. RESULTS Using the abovementioned technique, no filling defect was seen if a vein was occluded near its entrance into the sinus (N = 8) or at a single point over the cortex (N = 7). If a longer trajectory (10-14 mm.) was obstructed, a clear staining defect was seen in 13 out of 16 hemispheres; the three remaining cases seemed to be technical failures. CONCLUSION: A new method is described to investigate the draining area of a cerebral vein. Although the validity of the method is proven in an animal model, it seems a good technique for investigation of human brains postmortem. Application of this technique will contribute to the understanding of the pathophysiology of venous diseases and also elucidate the role of the venous anastomotic pathways.

Identification of subgroups of high-grade oligodendroglial tumors by comparative genomic hybridization.

In contrast to astrocytic tumors, approximately two thirds of anaplastic oligodendrogliomas are reported to be chemosensitive. Relatively little is known about the genetic aberrations in oligodendroglial tumors (OTs). In order to elucidate oligodendroglial oncogenesis and to find specific genetic aberrations that may have prognostic and therapeutic implications, we performed comparative genomic hybridization (CGH) to detect chromosomal copy number changes in 17 low-grade OTs (LG-OTs) and 12 high-grade OTs (HG-OTs) lacking a prominent astrocytic component. Loss of chromosome 1p (79%) and 19q (76%) were most frequently detected by CGH, all LG-OTs and 50% of the HG-OTs contained -1p (including 1p36-32), -19q (including 19q13.3), or both, and the rest of the HG-OTs showed +7, -10, or both. Since losses of 1p36-32 and 19q13.3 were mutually exclusive with +7 or -10, the HG-OTs could be divided in -1p/-19q and +7/-10 tumors. While the -1p/-19q tumors can be considered as pure anaplastic oligodendrogliomas, the +7/-10 tumors may rather be glioblastomas with prominent oligodendroglial differentiation. We conclude that CGH is a powerful tool to assist in the identification of 2 major subgroups of HG-OTs with prognostic and possibly therapeutic relevance.