Tejas Sankar

Virtual glioblastoma: growth, migration and treatment in a three-dimensional mathematical model

Objectives:  Glioblastomas are aggressive primary brain cancers that are characterized by extensive infiltration into the brain and are highly resistant to treatment. Through mathematical modelling, we model the process of invasion and predict the relative importance of mechanisms contributing to malignant invasion. Clinically, we predict patterns of tumour recurrence following various modes of therapeutic intervention.

Miniaturized handheld confocal microscopy for neurosurgery: results in an experimental glioblastoma model.

INTRODUCTIONRecent developments in optical science and image processing have miniaturized the components required for confocal microscopy. Clinical confocal imaging applications have emerged, including assessment of colonic mucosal dysplasia during colonoscopy. We present our initial experience with handheld, miniaturized confocal imaging in a murine brain tumor model. METHODSTwelve C57/BL6 mice were implanted intracranially with 105 GL261 glioblastoma cells. The brains of 6 anesthetized mice each at 14 and 21 days after implantation were exposed surgically, and the brain surface was imaged using a handheld confocal probe affixed to a stereotactic frame. The probe was moved systematically over regions of normal and tumor-containing tissue. Intravenous fluorescein and topical acriflavine contrast agents were used. Biopsies were obtained at each imaging site beneath the probe and assessed histologically. Mice were killed after imaging. RESULTSHandheld confocal imaging produced exquisite images, well-correlated with corresponding histologic sections, of cellular shape and tissue architecture in murine brain infiltrated by glial neoplasm. Reproducible patterns of cortical vasculature, as well as normal gray and white matter, were identified. Imaging effectively distinguished between tumor and nontumor tissue, including infiltrative tumor margins. Margins were easily identified by observers without prior neuropathology training after minimum experience with the technology. CONCLUSIONMiniaturized handheld confocal imaging may assist neurosurgeons in detecting infiltrative brain tumor margins during surgery. It may help to avoid sampling error during biopsy of heterogeneous glial neoplasms, with the potential to supplement conventional intraoperative frozen section pathology. Clinical trials are warranted on the basis of these promising initial results.

Monocyte galactose/N-acetylgalactosamine-specific C-type lectin receptor stimulant immunotherapy of an experimental glioma. Part 1: stimulatory effects on blood monocytes and monocyte-derived cells of the brain

Objectives Immunotherapy with immunostimulants is an attractive therapy against gliomas. C-type lectin receptors specific for galactose/N-acetylgalactosamine (GCLR) regulate cellular differentiation, recognition, and trafficking of monocyte-derived cells. A peptide mimetic of GCLR ligands (GCLRP) was used to activate blood monocytes and populations of myeloid-derived cells against a murine glioblastoma. Methods The ability of GCLRP to stimulate phagocytosis by human microglia and monocyte-derived cells of the brain (MDCB) isolated from a human glioblastoma was initially assessed in vitro. Induction of activation markers on blood monocytes was assayed by flow cytometry after administration of GCLRP to naive mice. C57BL/6 mice underwent stereotactic intracranial implantation of GL261 glioma cells and were randomized for tumor size by magnetic resonance imaging, which was also used to assess increase in tumor size. Brain tumor tissues were analyzed using flow cytometry, histology, and enzyme-linked immunosorbent assay with respect to tumor, peritumoral area, and contralateral hemisphere regions. Results GCLRP exhibited strong stimulatory effect on MDCBs and blood monocytes in vitro and in vivo. GCLRP was associated with an increased percentage of precursors of dendritic cells in the blood (P = 0.003), which differentiated into patrolling macrophages in tumoral (P = 0.001) and peritumoral areas (P = 0.04), rather than into dendritic cells, as in control animals. Treatment with GCLRP did not result in a significant change in survival of mice bearing a tumor. Conclusions In vitro and in vivo activation of monocytes was achieved by administration of GCLR to mice. GCLRP-activated blood monocytes were recruited to the brain and exhibited specific phenotypes corresponding with tumor region (glioma, peritumoral zone, and contralateral glioma-free hemisphere). GCLRP treatment alone was associated with increased glioma mass as the result of the infiltration of phagocytic cells. Regional specificity for MDCB may have significant tumor treatment implications.

Magnetic resonance imaging volumetric assessment of the extent of contrast enhancement and resection in oligodendroglial tumors

OBJECT Oligodendrogliomas that enhance on MR images are associated with poor prognosis. However, the importance of the volume of enhancing tumor tissue, and the extent of its resection, is uncertain. The authors examined the prognostic significance of preoperative and residual postoperative enhancing tissue volumes in a large single-center series of patients with oligodendroglioma. They also examined the relationship between enhancement and characteristic genetic signatures in oligodendroglial tumors, specifically deletion of 1p and 19q (del 1p/19q). METHODS The authors retrospectively analyzed 100 consecutive cases of oligodendroglioma involving patients who had undergone T1-weighted gadolinium-enhanced MRI at diagnosis and immediately after initial surgical intervention. The presence of preoperative enhancement was determined by consensus. Preoperative and residual postoperative volumes were measured using a quantitative, semiautomated method by a single blinded observer. Intrarater reliability for preoperative volumes was confirmed by remeasurement in a subset of patients 3 months later. Intrarater and interrater reliability for residual postoperative volumes was confirmed by remeasurement of these volumes by both the original and a second blinded observer. Multivariate analysis was used to assess the influence of contrast enhancement at diagnosis and the volume of pre- and postoperative contrast-enhancing tumor tissue on time to relapse (TTR) and overall survival (OS), while controlling for confounding clinical, pathological, and genetic factors. RESULTS Sixty-three of 100 patients had enhancing tumors at initial presentation. Presence of contrast enhancement at diagnosis was related to reduced TTR and OS on univariate analysis but was not significantly related on multivariate analysis. In enhancing tumors, however, greater initial volume of enhancing tissue correlated with shortened TTR (p = 0.00070). Reduced postoperative residual enhancing volume and a relatively greater resection of enhancing tissue correlated with longer OS (p = 0.0012 and 0.0041, respectively). Interestingly, patients in whom 100% of enhancing tumor was resected had significantly longer TTR (174 vs 64 weeks) and OS (392 vs 135 weeks) than those with any residual enhancing tumor postoperatively. This prognostic benefit was not consistently maintained with greater than 90% or even greater than 95% resection of enhancing tissue. There was no relationship between presence or volume of enhancement and del 1p/19q. CONCLUSIONS In enhancing oligodendrogliomas, completely resecting enhancing tissue independently improves outcome, irrespective of histological grade or genetic status. This finding supports aggressive resection and may impact treatment planning for patients with these tumors.

Activated autologous macrophage implantation in a large-animal model of spinal cord injury.

OBJECT Axonal regeneration may be hindered following spinal cord injury (SCI) by a limited immune response and insufficient macrophage recruitment. This limitation has been partially surmounted in small-mammal models of SCI by implanting activated autologous macrophages (AAMs). The authors sought to replicate these results in a canine model of partial SCI. METHODS Six dogs underwent left T-13 spinal cord hemisection. The AAMs were implanted at both ends of the lesion in 4 dogs, and 2 other dogs received sham implantations of cell media. Cortical motor evoked potentials (MEPs) were used to assess electrophysiological recovery. Functional motor recovery was assessed with a modified Tarlov Scale. After 9 months, animals were injected with wheat germ agglutinin-horseradish peroxidase at L-2 and killed for histological assessment. RESULTS Three of the 4 dogs that received AAM implants and 1 of the 2 negative control dogs showed clear recovery of MEP response. Behavioral assessment showed no difference in motor function between the AAM-treated and control groups. Histological investigation with an axonal retrograde tracer showed neither local fiber crossing nor significant uptake in the contralateral red nucleus in both implanted and negative control groups. CONCLUSIONS In a large-animal model of partial SCI treated with implanted AAMs, the authors saw no morphological or histological evidence of axonal regeneration. Although they observed partial electrophysiological and functional motor recovery in all dogs, this recovery was not enhanced in animals treated with implanted AAMs. Furthermore, there was no morphological or histological evidence of axonal regeneration in animals with implants that accounted for the observed recovery. The explanation for this finding is probably multifactorial, but the authors believe that the AAM implantation does not produce axonal regeneration, and therefore is a technology that requires further investigation before it can be clinically relied on to ameliorate SCI.

Neuromodulation for treatment-refractory major depressive disorder

Major depressive disorder is among the most prevalent psychiatric disorders and is a leading cause of morbidity and lost productivity.[1][1] The 1-year prevalence of major depressive disorder in the Canadian population is 3.2%–4.6%.[2][2] A large, multisite prospective trial showed that only 28%

Intracranial Implantation with Subsequent 3D In Vivo Bioluminescent Imaging of Murine Gliomas

The mouse glioma 261 (GL261) is recognized as an in vivo model system that recapitulates many of the features of human glioblastoma multiforme (GBM). The cell line was originally induced by intracranial injection of 3-methyl-cholantrene into a C57BL/6 syngeneic mouse strain (1); therefore, immunologically competent C57BL/6 mice can be used. While we use GL261, the following protocol can be used for the implantation and monitoring of any intracranial mouse tumor model. GL261 cells were engineered to stably express firefly luciferase (GL261-luc). We also created the brighter GL261-luc2 cell line by stable transfection of the luc2 gene expressed from the CMV promoter. C57BL/6-cBrd/cBrd/Cr mice (albino variant of C57BL/6) from the National Cancer Institute, Frederick, MD were used to eliminate the light attenuation caused by black skin and fur. With the use of albino C57BL/6 mice; in vivo imaging using the IVIS Spectrum in vivo imaging system is possible from the day of implantation (Caliper Life Sciences, Hopkinton, MA). The GL261-luc and GL261-luc2 cell lines showed the same in vivo behavior as the parental GL261 cells. Some of the shared histological features present in human GBMs and this mouse model include: tumor necrosis, pseudopalisades, neovascularization, invasion, hypercellularity, and inflammation (1). Prior to implantation animals were anesthetized by an intraperitoneal injection of ketamine (50 mg/kg), xylazine (5 mg/kg) and buprenorphine (0.05 mg/kg), placed in a stereotactic apparatus and an incision was made with a scalpel over the cranial midline. A burrhole was made 0.1 mm posterior to the bregma and 2.3mm to the right of the midline. A needle was inserted to a depth of 3mm and withdrawn 0.4 mm to a depth of 2.6 mm. Two μl of GL261-luc or GL261-luc2 cells (10(7) cells/ml) were infused over the course of 3 minutes. The burrhole was closed with bonewax and the incision was sutured. Following stereotactic implantation the bioluminescent cells are detectable from the day of implantation and the tumor can be analyzed using the 3D image reconstruction feature of the IVIS Spectrum instrument. Animals receive a subcutaneous injection of 150 μg luciferin /kg body weight 20 min prior to imaging. Tumor burden is quantified using mean tumor bioluminescence over time. Tumor-bearing mice were observed daily to assess morbidity and were euthanized when one or more of the following symptoms are present: lethargy, failure to ambulate, hunched posture, failure to groom, anorexia resulting in >10% loss of weight. Tumors were evident in all of the animals on necropsy.

Neurosurgical implications of mannitol accumulation within a meningioma and its peritumoral region demonstrated by magnetic resonance spectroscopy: Case report

Mannitol is widely considered the hyperosmolar therapy of choice in routine neurosurgical practice for the reduction of intracranial pressure (ICP). The authors present a unique case of a patient with a large meningioma treated with mannitol, in which mannitol accumulation within the tumor and its surrounding parenchyma was shown using in vivo magnetic resonance spectroscopy (MRS). This rare appearance of mannitol on MRS was characterized by a wide-based peak at 3.8 ppm, which remained detectable several hours after the last dose. These findings provide the first in vivo evidence in support of the prevailing theory that mannitol leakage into the peritumoral edematous region may contribute to rebound increases in ICP and suggest that this phenomenon has the potential to occur in extraaxial tumors. Judicious use of mannitol in the setting of elevated ICP due to tumor may be indicated to avoid potentially deleterious side effects caused by its accumulation.

Monocyte galactose/N-acetylgalactosamine-specific C-type lectin receptor stimulant immunotherapy of an experimental glioma. Part II: combination with external radiation improves survival.

Background A peptide mimetic of a ligand for the galactose/N-acetylgalactosamine-specific C-type lectin receptors (GCLR) exhibited monocyte-stimulating activity, but did not extend survival when applied alone against a syngeneic murine malignant glioma. In this study, the combined effect of GCLRP with radiation was investigated. Methods C57BL/6 mice underwent stereotactic intracranial implantation of GL261 glioma cells. Animals were grouped based on randomized tumor size by magnetic resonance imaging on day seven. One group that received cranial radiation (4 Gy on days seven and nine) only were compared with animals treated with radiation and GCLRP (4 Gy on days seven and nine combined with subcutaneous injection of 1 nmol/g on alternative days beginning on day seven). Magnetic resonance imaging was used to assess tumor growth and correlated with survival rate. Blood and brain tissues were analyzed with regard to tumor and contralateral hemisphere using fluorescence-activated cell sorting analysis, histology, and enzyme-linked immunosorbent assay. Results GCLRP activated peripheral monocytes and was associated with increased blood precursors of dendritic cells. Mean survival increased (P < 0.001) and tumor size was smaller (P < 0.02) in the GCLRP + radiation group compared to the radiation-only group. Accumulation of dendritic cells in both the tumoral hemisphere (P < 0.005) and contralateral tumor-free hemisphere (P < 0.01) was associated with treatment. Conclusion Specific populations of monocyte-derived brain cells develop critical relationships with malignant gliomas. The biological effect of GCLRP in combination with radiation may be more successful because of the damage incurred by tumor cells by radiation and the enhanced or preserved presentation of tumor cell antigens by GCLRP-activated immune cells. Monocyte-derived brain cells may be important targets for creating effective immunological modalities such as employing the receptor system described in this study.

Brain retraction and thickness of cerebral neocortex: An automated technique for detecting retraction-induced anatomic changes using magnetic resonance imaging

BACKGROUND Treating deep-seated cerebral lesions often requires retracting the brain. Retraction, however, causes clinically significant postoperative neurological deficits in 3% to 9% of intracranial cases. OBJECTIVE This pilot study used automated analysis of postoperative magnetic resonance images (MRIs) to determine whether brain retraction caused local anatomic changes to the cerebral neocortex and whether such changes represented sensitive markers for detecting brain retraction injury. METHODS Pre- and postoperative maps of whole-brain cortical thickness were generated from 3-dimensional MRIs of 6 patients who underwent selective amygdalohippocampectomy for temporal lobe epilepsy (5 left hemispheres, 1 right hemisphere). Mean cortical thickness was determined in the inferior temporal gyrus (ITG test), where a retractor was placed during surgery, and in 2 control gyri—the posterior portion of the inferior temporal gyrus (ITG control) and motor cortex control. Regions of cortical thinning were also compared with signs of retraction injury on early postoperative MRIs. RESULTS Postoperative maps of cortical thickness showed thinning in the inferior temporal gyrus where the retractor was placed in 5 patients. Postoperatively, mean cortical thickness declined from 4.1 ± 0.4 mm to 2.9 ± 0.9 mm in ITG test (P = .03) and was unchanged in the control regions. Anatomically, the region of neocortical thinning correlated with postoperative edema on MRIs obtained within 48 hours of surgery. CONCLUSION Postoperative MRIs can be successfully interrogated for information on cortical thickness. Brain retraction is associated with chronic local thinning of the neocortex. This automated technique may be sensitive enough to detect regions at risk for functional impairment during craniotomy that cannot be easily detected on postoperative structural imaging.