Multi-photon excitation fluorescence microscopy of brain-tumour tissue and analysis of cell density.

BACKGROUND: Recent studies have highlighted the importance of the complete resection of a brain tumour but the task often remains a challenge for the neurosurgeon. New technologies which add objective information beyond visualisation provided by the traditional operating microscope are required. In this study, we have analysed the cellular density of the tumour/brain interface using three dimensional multi-photon microscopy intensity-images of experimental gliomas and human brain-tumour biopsy samples. METHODS: The density of cellular nuclei was determined in specimens of experimental gliomas in a mouse model and human brain tumour biopsies by analysis of optical tissue sections. Three dimensional multi-photon microscopy image stacks were compared to serial H&E stained sections of conventional histopathology. FINDINGS: Both techniques consistently showed a good correlation of cell density values in solid tumour tissue of experimental gliomas versus adjacent brain. The multi-photon microscopy analysis of human biopsy specimens showed that optical analysis of native tissue provided information on the cellular density. CONCLUSIONS: Multi-photon microscopy is an efficient and rapid tool for the study of brain and brain tumour tissue. Multi-photon microscopy allows the detection of individual tumour cells and tumour cell clusters in native tissue biopsies and may therefore provide a tool in the identification of highly cellular lesions during the resection of brain tumours.

Lumbosacral glioblastoma and leptomeningeal gliomatosis complicating the course of a cervicothoracic astrocytoma WHO grade II.

CASE REPORT: The reported female patient underwent sub-total resection of an intra-medullary cervicothoracic astrocytoma classified as WHO grade II in 1984 at the age of 18 months and received local irradiation. In 1989, a local recurrence was diagnosed and a partial resection was performed. Sixteen years later, a small recurrent cervicothoracic tumour was found and spinal seeding to the equine nerve roots and the left cerebellar cortex was apparent on MRI. The patient was implanted with a ventriculoperitoneal shunt for a pseudo-tumour cerebri producing papilloedema, which eventually lead to amaurosis. After an extended biopsy, the invasive lumbosacral tumour was classified as glioblastoma multiforme. Two months later, the patient died after rapid progression of the caudal cranial nerve dysfunction. DISCUSSION AND CONCLUSION: Anaplastic progression and dissemination of spinal astrocytomas even two decades after initial diagnosis and treatment are rare. Therapies and diagnostic follow-up strategies are discussed.

Imaging of brain and brain tumor specimens by time-resolved multiphoton excitation microscopy ex vivo.

Multiphoton excitation fluorescent microscopy is a laser-based technology that allows subcellular resolution of native tissues in situ. We have recently applied this technology to the structural and photochemical imaging of cultured glioma cells and experimental gliomas ex vivo. We demonstrated that high microanatomical definition of the tumor, invasion zone, and normal adjacent brain can be obtained down to single-cell resolution in unprocessed tissue blocks. In this study, we used multiphoton excitation and four-dimensional microscopy to generate fluorescence lifetime maps of the murine brain anatomy, experimental glioma tissue, and biopsy specimens of human glial tumors. In murine brain, cellular and noncellular elements of the normal anatomy were identified. Distinct excitation profiles and lifetimes of endogenous fluorophores were identified for specific brain regions. Intracranial grafts of human glioma cell lines in mouse brain were used to study the excitation profiles and fluorescence lifetimes of tumor cells and adjacent host brain. These studies demonstrated that normal brain and tumor could be distinguished on the basis of fluorescence intensity and fluorescence lifetime profiles. Human brain specimens and brain tumor biopsies were also analyzed by multiphoton microscopy, which demonstrated distinct excitation and lifetime profiles in glioma specimens and tumor-adjacent brain. This study demonstrates that multiphoton excitation of autofluorescence can distinguish tumor tissue and normal brain based on the intensity and lifetime of fluorescence. Further technical developments in this technology may provide a means for in situ tissue analysis, which might be used to detect residual tumor at the resection edge.

Multiphoton excitation of autofluorescence for microscopy of glioma tissue.

OBJECTIVE: Intraoperative detection of residual tumor tissue in glioma surgery remains an important challenge because the extent of tumor removal is related to the prognosis of the disease. Multiphoton excited fluorescence tomography of living tissues provides high-resolution structural and photochemical imaging at a subcellular level. In this conceptual study, we have used multiphoton microscopy and fluorescence lifetime imaging (4D microscopy) to image cultured glioma cell lines, solid tumor, and invasive tumor cells in an experimental mouse glioma model and human glioma biopsy specimens. MATERIAL AND METHODS: A laser imaging system containing a mode-locked 80 MHz titanium:sapphire laser with a tuning range of 710 to 920 nm, a scan unit, and a time correlated single photon counting board was used to generate autofluorescence intensity images and fluorescence lifetime images of cultured cell lines, experimental intracranial gliomas in mouse brain, and biopsies of human gliomas. RESULTS: Multiphoton microscopy of native tumor bearing brain provided structural images of the normal brain anatomy at a subcellular resolution. Solid tumor, the tumor-brain interface, and single invasive tumor cells could be visualized. Fluorescence lifetime imaging demonstrated significantly different decay of the fluorescent signal in tumor versus normal brain, allowing a clear definition of the tumor-brain interface based on this parameter. Distinct fluorescence lifetimes of endogenous fluorophores were found in different cellular compartments in cultured glioma cells. The analysis of the relationship between the laser excitation wavelength and the lifetime of excitable fluorophores demonstrated distinct profiles for cells of different histotypes. CONCLUSION: Multiphoton excited fluorescence of endogenous fluorophores allows structural imaging of tumor and central nervous system histo-architecture at a subcellular level. The analysis of the decay of the fluorescent signal within specific excitation volumes by fluorescent lifetime imaging discriminates glioma cells and normal brain, and the excitation/lifetime profiles may further allow differentiation of cellular histotypes. This technology provides a noninvasive optical tissue analysis that may potentially be applied to an intraoperative analysis of resection plains in tumor surgery.

Zebra sign: cerebellar bleeding pattern characteristic of cerebrospinal fluid loss. Case report.

Supratentorial subdural hematoma is a well-known complication following spinal interventions. Less often, spinal or supratentorial interventions cause remote cerebellar hemorrhage (RCH). The exact pathomechanism accounting for RCH remains unclear, but an interventional or postinterventional loss of cerebrospinal fluid (CSF) seems to be involved in almost all cases. Hemorrhage is often characterized by a typical, streaky bleeding pattern due to blood spreading in the cerebellar sulci. Three different cases featuring this bleeding pattern following spinal, supratentorial, and thoracic surgery are presented. Possible pathomechanisms leading to RCH are discussed. Based on data from the underlying cases and the reviewed literature, the authors concluded that this zebra-pattern hemorrhage seems to be typical in a postoperative loss of CSF, which should always be considered on presentation of this bleeding pattern.

Prenatal diagnosis of 'true tail' with cartilage content?

A human tail is a rare congenital anomaly with a prominent lesion from the lumbosacrococcygeal region. According to Dao and Netzky human tails are classified into 'true tails' and 'pseudotails'. True tails comprise only mesenchymal tissue (adipose, connective, muscle, nerve tissue, blood vessels, and cutis). They are presumed to be remnants of the embryologic tail. All other lumbosacrococcygeal protrusions are summarized as pseudotails. Superficially they may resemble true tails. They contain normal or abnormal tissue, e.g. cartilage, lipoma and glioma. We report a case of prenatal diagnosis of a human tail in association with omphalocele, hydrocephalus and antiphospholipid-antibody syndrome resulting in a severe fetal growth restriction. Due to cartilage content the appendage had to be classified as 'pseudotail'. However, anatomical position and the decrease of length observed by consecutive ultrasound examinations at 14 and 21 weeks of gestation was suggestive of delayed regression of a 'true tail'. Furthermore, the association of antiphospholipid-antibody syndrome with congenital malformations is discussed.