Sergiy V. Kushchayev, Morgan B. Giers, Doris Hom Eng, Nikolay L. Martirosyan, Jennifer M. Eschbacher, Martin M. Mortazavi, Nicholas Theodore, Alyssa Panitch and Mark C. Preul
Spinal cord injury occurs in 2 phases. The initial trauma is followed by inflammation that leads to fibrous scar tissue, glial scarring, and cavity formation. Scarring causes further axon death around and above the injury. A reduction in secondary injury could lead to functional improvement. In this study, hyaluronic acid (HA) hydrogels were implanted into the gap formed in the hemisected spinal cord of Sprague-Dawley rats in an attempt to attenuate damage and regenerate tissue.
A T-10 hemisection spinal cord injury was created in adult male Sprague-Dawley rats; the rats were assigned to a sham, control (phosphate-buffered saline), or HA hydrogel–treated group. One cohort of 23 animals was followed for 12 weeks and underwent weekly behavioral assessments. At 12 weeks, retrograde tracing was performed by injecting Fluoro-Gold in the left L-2 gray matter. At 14 weeks, the animals were killed. The volume of the lesion and the number of cells labeled from retrograde tracing were calculated. Animals in a separate cohort were killed at 8 or 16 weeks and perfused for immunohistochemical analysis and transmission electron microscopy. Samples were stained using H & E, neurofilament stain (neurons and axons), silver stain (disrupted axons), glial fibrillary acidic protein stain (astrocytes), and Iba1 stain (mononuclear cells).
The lesions were significantly smaller in size and there were more retrograde-labeled cells in the red nuclei of the HA hydrogel–treated rats than in those of the controls; however, the behavioral assessments revealed no differences between the groups. The immunohistochemical analyses revealed decreased fibrous scarring and increased retention of organized intact axonal tissue in the HA hydrogel–treated group. There was a decreased presence of inflammatory cells in the HA hydrogel–treated group. No axonal or neuronal regeneration was observed.
The results of these experiments show that HA hydrogel had a neuroprotective effect on the spinal cord by decreasing the magnitude of secondary injury after a lacerating spinal cord injury. Although regeneration and behavioral improvement were not observed, the reduction in disorganized scar tissue and the retention of neurons near and above the lesion are important for future regenerative efforts. In addition, this gel would be useful as the base substrate in the development of a more complex scaffold.
Nikolay L. Martirosyan, Jennifer M. Eschbacher, M. Yashar S. Kalani, Jay D. Turner, Evgenii Belykh, Robert F. Spetzler, Peter Nakaji and Mark C. Preul
This study evaluated the utility, specificity, and sensitivity of intraoperative confocal laser endomicroscopy (CLE) to provide diagnostic information during resection of human brain tumors.
CLE imaging was used in the resection of intracranial neoplasms in 74 consecutive patients (31 male; mean age 47.5 years; sequential 10-month study period). Intraoperative in vivo and ex vivo CLE was performed after intravenous injection of fluorescein sodium (FNa). Tissue samples from CLE imaging–matched areas were acquired for comparison with routine histological analysis (frozen and permanent sections). CLE images were classified as diagnostic or nondiagnostic. The specificities and sensitivities of CLE and frozen sections for gliomas and meningiomas were calculated using permanent histological sections as the standard.
CLE images were obtained for each patient. The mean duration of intraoperative CLE system use was 15.7 minutes (range 3–73 minutes). A total of 20,734 CLE images were correlated with 267 biopsy specimens (mean number of images/biopsy location, in vivo 84, ex vivo 70). CLE images were diagnostic for 45.98% in vivo and 52.97% ex vivo specimens. After initiation of CLE, an average of 14 in vivo images and 7 ex vivo images were acquired before identification of a first diagnostic image. CLE specificity and sensitivity were, respectively, 94% and 91% for gliomas and 93% and 97% for meningiomas.
CLE with FNa provided intraoperative histological information during brain tumor removal. Specificities and sensitivities of CLE for gliomas and meningiomas were comparable to those for frozen sections. These data suggest that CLE could allow the interactive identification of tumor areas, substantially improving intraoperative decisions during the resection of brain tumors.
Nikolay L. Martirosyan, Joseph Georges, Jennifer M. Eschbacher, Daniel D. Cavalcanti, Ali M. Elhadi, Mohammed G. Abdelwahab, Adrienne C. Scheck, Peter Nakaji, Robert F. Spetzler and Mark C. Preul
The authors sought to assess the feasibility of a handheld visible-wavelength confocal endomicroscope imaging system (Optiscan 5.1, Optiscan Pty., Ltd.) using a variety of rapid-acting fluorophores to provide histological information on gliomas, tumor margins, and normal brain in animal models.
Mice (n = 25) implanted with GL261 cells were used to image fluorescein sodium (FNa), 5-aminolevulinic acid (5-ALA), acridine orange (AO), acriflavine (AF), and cresyl violet (CV). A U251 glioma xenograft model in rats (n = 5) was used to image sulforhodamine 101 (SR101). A swine (n = 3) model with AO was used to identify confocal features of normal brain. Images of normal brain, obvious tumor, and peritumoral zones were collected using the handheld confocal endomicroscope. Histological samples were acquired through biopsies from matched imaging areas. Samples were visualized with a benchtop confocal microscope. Histopathological features in corresponding confocal images and photomicrographs of H & E–stained tissues were reviewed.
Fluorescence induced by FNa, 5-ALA, AO, AF, CV, and SR101 and detected with the confocal endomicroscope allowed interpretation of histological features. Confocal endomicroscopy revealed satellite tumor cells within peritumoral tissue, a definitive tumor border, and striking fluorescent cellular and subcellular structures. Fluorescence in various tumor regions correlated with standard histology and known tissue architecture. Characteristic features of different areas of normal brain were identified as well.
Confocal endomicroscopy provided rapid histological information precisely related to the site of microscopic imaging with imaging characteristics of cells related to the unique labeling features of the fluorophores. Although experimental with further clinical trial validation required, these data suggest that intraoperative confocal imaging can help to distinguish normal brain from tumor and tumor margin and may have application in improving intraoperative decisions during resection of brain tumors.