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, 7 (7), e41760

Confocal Laser Endomicroscopy for Diagnosis and Histomorphologic Imaging of Brain Tumors in Vivo

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Confocal Laser Endomicroscopy for Diagnosis and Histomorphologic Imaging of Brain Tumors in Vivo

Sebastian Foersch et al. PLoS One.

Abstract

Early detection and evaluation of brain tumors during surgery is crucial for accurate resection. Currently cryosections during surgery are regularly performed. Confocal laser endomicroscopy (CLE) is a novel technique permitting in vivo histologic imaging with miniaturized endoscopic probes at excellent resolution. Aim of the current study was to evaluate CLE for in vivo diagnosis in different types and models of intracranial neoplasia. In vivo histomorphology of healthy brains and two different C6 glioma cell line allografts was evaluated in rats. One cell line expressed EYFP, the other cell line was used for staining with fluorescent dyes (fluorescein, acriflavine, FITC-dextran and Indocyanine green). To evaluate future application in patients, fresh surgical resection specimen of human intracranial tumors (n = 15) were examined (glioblastoma multiforme, meningioma, craniopharyngioma, acoustic neurinoma, brain metastasis, medulloblastoma, epidermoid tumor). Healthy brain tissue adjacent to the samples served as control. CLE yielded high-quality histomorphology of normal brain tissue and tumors. Different fluorescent agents revealed distinct aspects of tissue and cell structure (nuclear pattern, axonal pathways, hemorrhages). CLE discrimination of neoplastic from healthy brain tissue was easy to perform based on tissue and cellular architecture and resemblance with histopathology was excellent. Confocal laser endomicroscopy allows immediate in vivo imaging of normal and neoplastic brain tissue at high resolution. The technology might be transferred to scientific and clinical application in neurosurgery and neuropathology. It may become helpful to screen for tumor free margins and to improve the surgical resection of malignant brain tumors, and opens the door to in vivo molecular imaging of tumors and other neurologic disorders.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Experimental setting.
A - In vivo confocal laser endomicroscopy in anesthetized, tumor-bearing wistar rats. The animal is positioned in a stereotactic frame, which is connected to the confocal endomicroscopic probe. Detail: the tip of the probe is gently brought in contact with the intracranial window and images of the tumor, the transition zone and healthy brain tissue are gathered. B - Ex vivo imaging of fresh surgical resection specimen of brain tumors using CLE. Samples were gathered intraoperatively and immediately transferred to the imaging facility. Detail: imaging after topical staining with acriflavine.
Figure 2
Figure 2. Healthy cerebellum of a Wistar rat.
A - In vivo CLE enabled to image the histology of different parts of the brain, e. g. the cerebellum. After topical application of acriflavine, the characteristic layers of can be observed and specific Purkinje cells (asterisk) with their dendritic tree (triangle) are stained. B - The resemblance to ex vivo histopathologic staining is substantial.
Figure 3
Figure 3. C6WT glioma allograft.
A - Macroscopic view of two intracranial windows. On the left side the transition zone between healthy and tumor tissue can be observed. The other side served as control. B-D - Vessel morphology after intravenous injection of FITC-dextrane. (B) Tumor tissue shows massive extravasation with erythrocytes and FITC-dextrane in the interstitial zone (also seen in H&E staining). (C & E) Normal vessels and capillaries reveal structural integrity and erythrocytes can be observed in their physiological flow. E-G - Transition zone between healthy brain tissue (cross) and C6WT glioma allograft (asterisk) displayed in vivo using CLE (G) and ex vivo with H & E staining (E, F). Both entities show their specific histomorphological aspect in either one procedure.
Figure 4
Figure 4. C6EYFP glioma allograft.
A - Image of the transition zone between healthy brain tissue (cross) and tumor tissue (asterisk) with endogenous fluorescence in vivo using CLE. No added fluorescence was used for intravital imaging. Where allograft tissue reveals a characteristic histomorphology, healthy brain shows no signal at all. B - Ex vivo bench top fluorescence microscopy with DAPI nuclei counterstaining. Again the transition zone can be observed. C - CLE shows cellular and subcellular details of the tumor cells: as cells of glia-origin appendices (triangle) and cell soma (arrow) can be distinguished. D, E - Detail of fluorescent tumor cells in ex vivo bench top fluorescence and in vivo CLE. High resemblance between both imaging modalities was found. - Scale bars c – e: 30 µm.
Figure 5
Figure 5. C6EYFP glioma allograft.
Invading tumor cells distant from the main tumor (asterisk) can be observed in vivo (A) and are confirmed by ex vivo fluorescence microscopy (B). No fluorescent dye was applied for imaging in vivo. Since no auto-fluorescence was observed, specific signal results from transfected tumor cells. Ex vivo, no further staining procedure was performed, except counter staining of the nuclei with DAPI. Fluorescent tumor cells are also seen distant from the main tumor (arrows).
Figure 6
Figure 6. Glioblastoma.
Schematic (A) and macroscopic (B) image of resection specimen. Attached to the tumorous tissue (pink), a small piece of healthy brain tissue was observed (purple). Panel C-F - Ex vivo CLE of a fresh surgical resection specimen of healthy brain tissue (C, D) in direct proximity of the glioblastoma specimen (E, F) of the same patient. Healthy brain tissue reveals neuron or microglial cells with appendices (arrows). In comparison to healthy brain tissue the tumor reveals rampant excessive growth with atypic nuclei and abnormal nuclear-to-cytoplasm ratio. Atypic mitoses can be observed. Panel G-J - Ex vivo histopathology confirms the findings and diagnosis found in CLE (healthy brain tissue G, H – glioblastoma I, J).
Figure 7
Figure 7. Fresh human brain tumor biopsy specimens.
A - Ex vivo CLE of a craniopharyngioma. Small nuclei aligned in disarranged formations such as strands or trabecular structures represent the squamous origin of this mostly benign tumor. No calcifications can be observed using CLE or ex vivo (B) indicating the papillar type of this neoplasia. B - H.&E. staining of this lesion shows high similarity to CLE. C - Confocal images of a fresh resection specimen of a brain metastasis of a primary ductal mamma carcinoma. This tumor origin is consistent with the histomorphologic aspect of the neoplastic cells. Differences in size and shape of both the cells and the nuclei can be observed. Prominent nucleoli are also characteristic for the metastatic tissue. D - An excellent resemblance of the endomicroscopic images and the histopathologic gold standard can be observed. E, F - Typical example of a number of meningiomas, using CLE (E) and ex vivo (F). Characteristic pallisade-shaped and round cell formations can be observed representing psammoma bodies (outline). Tumor cell nuclei however remain mostly of equal size and shape, indicating the tumor’s benign origin. Again ex vivo histopathology by H.&E. staining strongly resembles the CLE images. G - Confocal laser endomicroscopy of an acoustic neurinoma. Nuclei accumulation can be frequently seen, consistent with the histopathologic feature of Verocay bodies (outline). Tumor fibers can be observed representing Antoni A structures (I). H, J - All characteristics of acoustic neurinoma can also be found in conventional ex vivo histopathology. K - An epidermoid cyst tumor was subject to the last confocal laser endomicroscopic examination. In CLE a crystal like structure could be observed with almost no nuclear staining. These cells represent desquamating epithelial cells. Massive keratin accumulation can also be observed. L - H.&E. staining again confirms findings of CLE. Scale bars represent 100 µm.

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