SCIENCE TIMES
out to determine if there was a difference in overall functional outcome at 6 months between cohorts. Patients were stratified by country group and planned operation (craniotomy or other), and within these strata they were allocated according to a minimization algorithm based on age (,60 years, 60-69 years, or $70 years) and neurological deficit in the worst affected arm or leg (normal, weak, or paralyzed). Randomization to early surgery occurred in 307 of 601 patients, and 294 were randomized to initial conservative therapy. The 2 cohorts were similar in terms of both patient and ICH characteristics. The median age of patients was 65 years (range, 17-94) and 57% were males. Half of the patients in the early surgery group and 49% in the initial conservative treatment group had a Glasgow Coma Scale score (GCS) of 14 or 15 at randomization. The median volume of the ICH was 36 mL (range, 23-55) and the median depth from the cortex surface was 1 mm (range, 0-2). The planned method of evacuation in 98% of all cases was craniotomy. Patients were randomly assigned within 48 hours of ictus and a quarter of patients in both cohorts were assigned within 12 hours. Of the 301 assessable patients in the early surgery group, 96% had surgery and 4% did not have surgery because their families refused or they had other medical or logistical problems. Of 291 assessable patients in the initial conservative treatment group, 62 (21%) had surgery, of which decline in GCS, increased edema, rebleeding, and increased intracranial pressure were the most common (n ¼ 58) reasons for surgery. Post-randomization adverse events reported during the hospital stay before the 2-week point were similar in the 2 groups. Although patients receiving surgery had better discharge outcomes, there was no significant difference in the incidence of unfavorable 6-month outcome among those patients randomized to early surgery (59%) vs those randomized to initial conservative treatment (62%). Adjustment for the covariates age, GCS, hemorrhage volume, and neurological deficit made little difference. There was a trend toward decreased mortality in the surgical cohort at 6 months (18%) vs the initial conservative treatment group (24%), and the actual survival advantage during the first 6 months demonstrated a similar trend. As with many surgical trials, overall interpretation is somewhat limited due to cross over. Patients initially assigned to medical therapy with delayed deterioration that ultimately underwent surgery may have been saved from a fatal course, thus inflating the overall outcomes in the conservative cohort by intention to treat analysis. A further limitation is that a majority of patients in the trial were fully conscious or just
N12 | VOLUME 74 | NUMBER 2 | FEBRUARY 2014
confused. These patients may be optimally managed with conservative therapy with delayed surgery carried out only if they were to deteriorate. Thus, the trial did not directly determine the role of early surgery in patients with increased intracranial pressure, who are most likely to benefit from surgery. Additionally, when stratifying the patients into a good and bad prognosis cohort based on GCS and ICH size, those deemed to have a poor prognosis had a significantly better overall outcome in subgroup analysis. The average time from presentation to surgery was 26.7 hours, which may be too long to reverse permanent neurological damage and toxic ICH products. A trial may be necessary to demonstrate the potential benefits of surgery in close proximity to clinical decline. STICH II is the 15th randomized trial to assess the role of surgery in ICH as compared with medical therapy.7 Addition of STICH II to the 14 prior trials gives an overall sample size of 3,366 patients. Overall analysis demonstrates a beneficial role of surgery (OR ¼ 0.74; 95% CI, 0.64-0.86, P , .0001), but with significant heterogeneity due to differences in patient, ICH, and surgical characteristics. Cumulative assessment STICH II along with prior trials concerning of lobar ICH without intraventricular hemorrhage (n ¼ 923) demonstrates a trend toward a benefit of surgery (OR ¼ 0.78; 95% CI, 0.59-1.02, P ¼.07), but without significant heterogeneity. STICH II provides further evidence that certain patients with ICH may benefit from surgical therapy. Other less invasive surgical techniques may be more beneficial, including endoscopic-assisted ICH evacuation. Trials that employ stereotactic delivery of tissue plasminogen activator (tPA) to dissolve clots and tPA to dissolve intraventricular hemorrhages are currently underway.8-10 These minimal access techniques might be more beneficial for deep clots, intraventricular hemorrhage, and specific subsets of patients with superficial ICH. ROBERT M. STARKE RICARDO J. KOMOTAR E. SANDER CONNOLLY
REFERENCES 1. van Asch CJ, Luitse MJ, Rinkel GJ, van der Tweel I, Algra A, Klijn CJ. Incidence, case fatality, and functional outcome of intracerebral haemorrhage over time, according to age, sex, and ethnic origin: a systematic review and meta-analysis. Lancet Neurol. 2010;9(2):167-176. 2. Feigin VL, Lawes CM, Bennett DA, Anderson CS. Stroke epidemiology: a review of population-based studies of incidence, prevalence, and case-fatality in the late 20th century. Lancet Neurol. 2003;2(1): 43-53.
3. Siddique MS, Fernandes HM, Arene NU, Wooldridge TD, Fenwick JD, Mendelow AD. Changes in cerebral blood flow as measured by HMPAO SPECT in patients following spontaneous intracerebral haemorrhage. Acta Neurochir Suppl. 2000;76:517-520. 4. Mendelow AD. Mechanisms of ischemic brain damage with intracerebral hemorrhage. Stroke. 1993;24(suppl 12):I115-I117;; discussion I8-I9. 5. Prasad K, Mendelow AD, Gregson B. Surgery for primary supratentorial intracerebral haemorrhage. Cochrane Database Syst Rev. 2008;(4):CD000200. 6. Mendelow AD, Gregson BA, Fernandes HM, et al. Early surgery versus initial conservative treatment in patients with spontaneous supratentorial intracerebral haematomas in the International Surgical Trial in Intracerebral Haemorrhage (STICH): a randomised trial. Lancet. 2005;365(9457):387-397. 7. Mendelow AD, Gregson BA, Rowan EN, Murray GD, Gholkar A, Mitchell PM. Early surgery versus initial conservative treatment in patients with spontaneous supratentorial lobar intracerebral haematomas (STICH II): a randomised trial. Lancet. 2013;382(9890):397-408. 8. Hanley DF. Minimally Invasive Surgery plus rTPA for ICH Evacuation Phase III. 2013. Available at: http://braininjuryoutcomes.com/mistie-iii-about. Accessed November 11, 2013. 9. Naff N, Williams MA, Keyl PM, et al. Low-dose recombinant tissue-type plasminogen activator enhances clot resolution in brain hemorrhage: the intraventricular hemorrhage thrombolysis trial. Stroke. 2011;42(11):3009-3016. 10. Hanley DF. CLEAR-III clot lysis: evaluating accelerated resolution of intraventricular hemorrhage Phase III. 2013. Available at: http://braininjuryoutcomes.com/clear-about. Accessed November 11, 2013.
Detecting Brain Tumor With Raman Scattering Microscopy
I
nfiltrating gliomas are often difficult to distinguish from normal brain, leading to incomplete resections. Current strategies that label tumor cells suffer from inhomogeneous distribution within tumors, limited blood-brain barrier penetration, and other signal/noise and FDA approval limitations. Ji and colleagues recently reported a novel imaging technique using simulated Raman scattering (SRS) microscopy to differentiate healthy brain tissue from tumor-infiltrated brain tissue based on histoarchitectural and biochemical differences.1 SRS microscopy images biological tissues based on intrinsic components such as lipids, proteins, and DNA (Figures 1A and 1B). Different Raman spectra are seen in various brain regions due to macromolecule composition. The differential ratio of Raman signals at
www.neurosurgery-online.com
Copyright © Congress of Neurological Surgeons. Unauthorized reproduction of this article is prohibited.
SCIENCE TIMES
Figure 1. Two-color SRS microscopy. A, experimental setup of epi-SRS microscopy. Stokes beam was modulated at high frequency (10 MHz), and the weak SRL signal was demodulated by a lock-in amplifier. Epi-detection scheme was used for in vivo brain imaging and ex vivo imaging on fresh tissues. CS, coverslip; DC, dichroic mirror; EOM, electro-optical modulator; FI, optical filter; PD, photodiode; SL, saline. B, energy diagram of the SRS process, where pump and Stokes photons excite the ground-state (n ¼ 0) molecules to their vibrational excited state (n ¼ 1), resulting in the reduction of pump intensity– stimulated Raman loss (SRL) and the increase in Stokes intensity–stimulated Raman gain (SRG). C, Raman spectra from frozen sections of a mouse brain with human GBM xenografts show white matter, cortex, and tumor. The marked frequencies at 2845 and 2930 cm21 were chosen for 2-color SRS imaging. (From [Ji M, Orringer DA, Freudiger CW, et al. Rapid, label-free detection of brain tumors with stimulated Raman scattering microscopy. Sci Transl Med. 2013; 5(201):201ra119]. Reprinted with permission from AAAS).
2930 and 2845 cm21 (S2930/S2845) reflects the different lipid and protein concentrations of brain regions. Highly cellular regions/solid tumors had a mean intensity ratio of S2930/S2845 ¼ 4.0 6 0.3, whereas normal cortex had a mean ratio of 1.6 6 0.1, and white matter had a ratio of 0.93 6 0.04 (Figure 1C). The authors used SRS microscopy to detect normal tissue architecture of the brain areas like the hippocampal cornus ammonis 1 (CA1) region. Unlike dye-based technologies, SRS microscopy is a label-free technique. Furthermore, SRS microscopy has 3-D capabilities, making it ideal for immediate intraoperative imaging and potentially supplanting tissue sections for neuropathologic analysis. The authors showed that 2-color SRS microscopy can detect gliomas in an ex vivo GBM xenograft mouse model. Results obtained from this technique correlated with H&E findings commonly used for GBM diagnoses.
NEUROSURGERY
The greatest advantage of SRS microscopy is its ability to detect tumor margins that appear normal under standard bright-field conditions. Coronal brain sections of human GBM xenografts were imaged with SRS and H&E microscopy for comparison. Compared to H&E diagnosis given by 3 different neuropathologists, SRS microscopy was 98.7% accurate (74/75) in diagnosing normal tissues, 98.7% accurate (74/ 75) in diagnosing infiltrating gliomas, and 100% (75/75) accurate in diagnosing high-density gliomas. The authors also tested feasibility of in vivo SRS microscopy. GBM xenografts in mice were exposed using a cranial window for SRS imaging. Although tumor was not grossly visible on the brain surface, SRS microscopy successfully detected tumor (Figure 2) that was later verified with H&E stained sections acquired from a coronal plane perpendicular to the imaging plane. SRS microscopy was also
validated with fresh human GBM specimens and corresponded with H&E analysis. SRS microscopy cannot provide all the current architectural, genetic, and biochemical data available from tissue sectioning, but potentially permits real-time discrimination of glioma boundaries and normal brain, limited to only 100 mm of spatial resolution. Furthermore, SRS microscopy image quality is affected by respiratory and cardiac cycles. Future advances in applying this technology, such as via intraoperative handheld SRS microscopy, may permit intraoperative rapid detection of residual tumor at resection edges, thereby maximizing safe surgical resection and improving glioma patient outcomes. KELLI B. POINTER RAY R. ZHANG JOHN S. KUO ROBERT J. DEMPSEY
VOLUME 74 | NUMBER 2 | FEBRUARY 2014 | N13
Copyright © Congress of Neurological Surgeons. Unauthorized reproduction of this article is prohibited.
SCIENCE TIMES
Figure 2. In vivo SRS microscopy images of human GBM xenografts. Images are representative of 6 mice. SRS imaging was carried out via acute cranial window preparation in mice 24 days after implantation of human GBM xenografts. A, bright-field microscopy appears grossly normal, whereas SRS microscopy within the same FOV demonstrates distinctions between tumorinfiltrated areas and non-infiltrated brain (normal), with a normal brain–tumor interface (dashed line). B-D, high-magnification views within the tumor (B), at the tumor-brain interface (C), and within normal brain (D). (From [Ji M, Orringer DA, Freudiger CW, et al. Rapid, label-free detection of brain tumors with stimulated Raman scattering microscopy. Sci Transl Med. 2013;5 (201):201ra119]. Reprinted with permission from AAAS).
REFERENCE 1. Ji M, Orringer DA, Freudiger CW, et al. Rapid, label-free detection of brain tumors with stimulated Raman scattering microscopy. Sci Transl Med. 2013;5 (201):201ra119.
Knockdown of LGR5 Suppresses the Proliferation of Glioma Cells in Vitro and in Vivo
G
liomas are the most common primary brain tumor in adults and are categorized into 1 of 4 WHO grades based on histopathological characteristics.1 The aggressive, infiltrative nature of high-grade gliomas portends a grave prognosis despite aggressive chemotherapy, radiation, and surgical resection. Current efforts for developing novel and effective treatment options for gliomas have focused on molecular targets that interrupt tumor proliferation. However, these techniques require a thorough understanding of the cell signaling mechanisms of gliomas. Previous research has
N14 | VOLUME 74 | NUMBER 2 | FEBRUARY 2014
delineated a number of proteins critical for normal growth and development that includes regulation of ligand-mediated signaling, cell adhesion, and formation of the extracellular matrix for cell migration.2,3 The leucine-rich repeat containing G protein-coupled receptor 5 (LGR5), has been recently shown to play a key functional role in normal cellular development.4 When abnormal expression of LGR5 is present, tumorigenesis has been shown to occur through activation of the Wnt pathway. However, the exact mechanisms of LGR5 activity in glioma cells have not been elucidated. Recent research published in the October edition of Oncology Reports from Zhang et al in China have confirmed the expression of LGR5 in human gliomas and its correlation with pathological grade and proliferation.5 Additionally, they demonstrate compelling evidence of tumor growth inhibition using RNA interference against LGR5 in both in vitro and in vivo models. The investigators collected 54 gliomas of varying grade from patients who presented for initial surgical resection and had no prior treatments with radiation or chemotherapy. All of the specimens were subjected to immunohistochemical assays for LGR5. LGR5 immunoreactivity
scores (IRS), LGR5 proliferative indices (PI), and the percentage of Ki-67 positive cells were quantified. Both the LGR5 IRS and PI correlated with increasing WHO grade. The investigators then studied the LGR5 expression levels in 3 glioma cell lines (U118, U87, and U251) in vitro and compared them to LGR5 levels expressed in normal human astrocytes. The results demonstrated higher LGR5 expression in the glioma cell lines compared to the normal astrocytes. The team then investigated the role of LGR5 in the malignant transformation of gliomas. The U87 cell lines were transfected with RNAi against LGR5, which resulted in a significant and specific decrease in the endogenous expression of LGR5. In order to evaluate the effect of LGR5 on the growth of U87 cells, the researchers conducted viability assays using U87, U87negative control (NC), and U87-knockdown (KD) cells. Their data showed a marked growth inhibition of U87-KD cells compared to the other groups. Additionally, there were no significant differences in cell growth between the U87 and U87-NC cells. Knockdown of LGR5 also decreased the number and size of colony formation in U87-KD cells
www.neurosurgery-online.com
Copyright © Congress of Neurological Surgeons. Unauthorized reproduction of this article is prohibited.
out to determine if there was a difference in overall functional outcome at 6 months between cohorts. Patients were stratified by country group and planned operation (craniotomy or other), and within these strata they were allocated according to a minimization algorithm based on age (,60 years, 60-69 years, or $70 years) and neurological deficit in the worst affected arm or leg (normal, weak, or paralyzed). Randomization to early surgery occurred in 307 of 601 patients, and 294 were randomized to initial conservative therapy. The 2 cohorts were similar in terms of both patient and ICH characteristics. The median age of patients was 65 years (range, 17-94) and 57% were males. Half of the patients in the early surgery group and 49% in the initial conservative treatment group had a Glasgow Coma Scale score (GCS) of 14 or 15 at randomization. The median volume of the ICH was 36 mL (range, 23-55) and the median depth from the cortex surface was 1 mm (range, 0-2). The planned method of evacuation in 98% of all cases was craniotomy. Patients were randomly assigned within 48 hours of ictus and a quarter of patients in both cohorts were assigned within 12 hours. Of the 301 assessable patients in the early surgery group, 96% had surgery and 4% did not have surgery because their families refused or they had other medical or logistical problems. Of 291 assessable patients in the initial conservative treatment group, 62 (21%) had surgery, of which decline in GCS, increased edema, rebleeding, and increased intracranial pressure were the most common (n ¼ 58) reasons for surgery. Post-randomization adverse events reported during the hospital stay before the 2-week point were similar in the 2 groups. Although patients receiving surgery had better discharge outcomes, there was no significant difference in the incidence of unfavorable 6-month outcome among those patients randomized to early surgery (59%) vs those randomized to initial conservative treatment (62%). Adjustment for the covariates age, GCS, hemorrhage volume, and neurological deficit made little difference. There was a trend toward decreased mortality in the surgical cohort at 6 months (18%) vs the initial conservative treatment group (24%), and the actual survival advantage during the first 6 months demonstrated a similar trend. As with many surgical trials, overall interpretation is somewhat limited due to cross over. Patients initially assigned to medical therapy with delayed deterioration that ultimately underwent surgery may have been saved from a fatal course, thus inflating the overall outcomes in the conservative cohort by intention to treat analysis. A further limitation is that a majority of patients in the trial were fully conscious or just
N12 | VOLUME 74 | NUMBER 2 | FEBRUARY 2014
confused. These patients may be optimally managed with conservative therapy with delayed surgery carried out only if they were to deteriorate. Thus, the trial did not directly determine the role of early surgery in patients with increased intracranial pressure, who are most likely to benefit from surgery. Additionally, when stratifying the patients into a good and bad prognosis cohort based on GCS and ICH size, those deemed to have a poor prognosis had a significantly better overall outcome in subgroup analysis. The average time from presentation to surgery was 26.7 hours, which may be too long to reverse permanent neurological damage and toxic ICH products. A trial may be necessary to demonstrate the potential benefits of surgery in close proximity to clinical decline. STICH II is the 15th randomized trial to assess the role of surgery in ICH as compared with medical therapy.7 Addition of STICH II to the 14 prior trials gives an overall sample size of 3,366 patients. Overall analysis demonstrates a beneficial role of surgery (OR ¼ 0.74; 95% CI, 0.64-0.86, P , .0001), but with significant heterogeneity due to differences in patient, ICH, and surgical characteristics. Cumulative assessment STICH II along with prior trials concerning of lobar ICH without intraventricular hemorrhage (n ¼ 923) demonstrates a trend toward a benefit of surgery (OR ¼ 0.78; 95% CI, 0.59-1.02, P ¼.07), but without significant heterogeneity. STICH II provides further evidence that certain patients with ICH may benefit from surgical therapy. Other less invasive surgical techniques may be more beneficial, including endoscopic-assisted ICH evacuation. Trials that employ stereotactic delivery of tissue plasminogen activator (tPA) to dissolve clots and tPA to dissolve intraventricular hemorrhages are currently underway.8-10 These minimal access techniques might be more beneficial for deep clots, intraventricular hemorrhage, and specific subsets of patients with superficial ICH. ROBERT M. STARKE RICARDO J. KOMOTAR E. SANDER CONNOLLY
REFERENCES 1. van Asch CJ, Luitse MJ, Rinkel GJ, van der Tweel I, Algra A, Klijn CJ. Incidence, case fatality, and functional outcome of intracerebral haemorrhage over time, according to age, sex, and ethnic origin: a systematic review and meta-analysis. Lancet Neurol. 2010;9(2):167-176. 2. Feigin VL, Lawes CM, Bennett DA, Anderson CS. Stroke epidemiology: a review of population-based studies of incidence, prevalence, and case-fatality in the late 20th century. Lancet Neurol. 2003;2(1): 43-53.
3. Siddique MS, Fernandes HM, Arene NU, Wooldridge TD, Fenwick JD, Mendelow AD. Changes in cerebral blood flow as measured by HMPAO SPECT in patients following spontaneous intracerebral haemorrhage. Acta Neurochir Suppl. 2000;76:517-520. 4. Mendelow AD. Mechanisms of ischemic brain damage with intracerebral hemorrhage. Stroke. 1993;24(suppl 12):I115-I117;; discussion I8-I9. 5. Prasad K, Mendelow AD, Gregson B. Surgery for primary supratentorial intracerebral haemorrhage. Cochrane Database Syst Rev. 2008;(4):CD000200. 6. Mendelow AD, Gregson BA, Fernandes HM, et al. Early surgery versus initial conservative treatment in patients with spontaneous supratentorial intracerebral haematomas in the International Surgical Trial in Intracerebral Haemorrhage (STICH): a randomised trial. Lancet. 2005;365(9457):387-397. 7. Mendelow AD, Gregson BA, Rowan EN, Murray GD, Gholkar A, Mitchell PM. Early surgery versus initial conservative treatment in patients with spontaneous supratentorial lobar intracerebral haematomas (STICH II): a randomised trial. Lancet. 2013;382(9890):397-408. 8. Hanley DF. Minimally Invasive Surgery plus rTPA for ICH Evacuation Phase III. 2013. Available at: http://braininjuryoutcomes.com/mistie-iii-about. Accessed November 11, 2013. 9. Naff N, Williams MA, Keyl PM, et al. Low-dose recombinant tissue-type plasminogen activator enhances clot resolution in brain hemorrhage: the intraventricular hemorrhage thrombolysis trial. Stroke. 2011;42(11):3009-3016. 10. Hanley DF. CLEAR-III clot lysis: evaluating accelerated resolution of intraventricular hemorrhage Phase III. 2013. Available at: http://braininjuryoutcomes.com/clear-about. Accessed November 11, 2013.
Detecting Brain Tumor With Raman Scattering Microscopy
I
nfiltrating gliomas are often difficult to distinguish from normal brain, leading to incomplete resections. Current strategies that label tumor cells suffer from inhomogeneous distribution within tumors, limited blood-brain barrier penetration, and other signal/noise and FDA approval limitations. Ji and colleagues recently reported a novel imaging technique using simulated Raman scattering (SRS) microscopy to differentiate healthy brain tissue from tumor-infiltrated brain tissue based on histoarchitectural and biochemical differences.1 SRS microscopy images biological tissues based on intrinsic components such as lipids, proteins, and DNA (Figures 1A and 1B). Different Raman spectra are seen in various brain regions due to macromolecule composition. The differential ratio of Raman signals at
www.neurosurgery-online.com
Copyright © Congress of Neurological Surgeons. Unauthorized reproduction of this article is prohibited.
SCIENCE TIMES
Figure 1. Two-color SRS microscopy. A, experimental setup of epi-SRS microscopy. Stokes beam was modulated at high frequency (10 MHz), and the weak SRL signal was demodulated by a lock-in amplifier. Epi-detection scheme was used for in vivo brain imaging and ex vivo imaging on fresh tissues. CS, coverslip; DC, dichroic mirror; EOM, electro-optical modulator; FI, optical filter; PD, photodiode; SL, saline. B, energy diagram of the SRS process, where pump and Stokes photons excite the ground-state (n ¼ 0) molecules to their vibrational excited state (n ¼ 1), resulting in the reduction of pump intensity– stimulated Raman loss (SRL) and the increase in Stokes intensity–stimulated Raman gain (SRG). C, Raman spectra from frozen sections of a mouse brain with human GBM xenografts show white matter, cortex, and tumor. The marked frequencies at 2845 and 2930 cm21 were chosen for 2-color SRS imaging. (From [Ji M, Orringer DA, Freudiger CW, et al. Rapid, label-free detection of brain tumors with stimulated Raman scattering microscopy. Sci Transl Med. 2013; 5(201):201ra119]. Reprinted with permission from AAAS).
2930 and 2845 cm21 (S2930/S2845) reflects the different lipid and protein concentrations of brain regions. Highly cellular regions/solid tumors had a mean intensity ratio of S2930/S2845 ¼ 4.0 6 0.3, whereas normal cortex had a mean ratio of 1.6 6 0.1, and white matter had a ratio of 0.93 6 0.04 (Figure 1C). The authors used SRS microscopy to detect normal tissue architecture of the brain areas like the hippocampal cornus ammonis 1 (CA1) region. Unlike dye-based technologies, SRS microscopy is a label-free technique. Furthermore, SRS microscopy has 3-D capabilities, making it ideal for immediate intraoperative imaging and potentially supplanting tissue sections for neuropathologic analysis. The authors showed that 2-color SRS microscopy can detect gliomas in an ex vivo GBM xenograft mouse model. Results obtained from this technique correlated with H&E findings commonly used for GBM diagnoses.
NEUROSURGERY
The greatest advantage of SRS microscopy is its ability to detect tumor margins that appear normal under standard bright-field conditions. Coronal brain sections of human GBM xenografts were imaged with SRS and H&E microscopy for comparison. Compared to H&E diagnosis given by 3 different neuropathologists, SRS microscopy was 98.7% accurate (74/75) in diagnosing normal tissues, 98.7% accurate (74/ 75) in diagnosing infiltrating gliomas, and 100% (75/75) accurate in diagnosing high-density gliomas. The authors also tested feasibility of in vivo SRS microscopy. GBM xenografts in mice were exposed using a cranial window for SRS imaging. Although tumor was not grossly visible on the brain surface, SRS microscopy successfully detected tumor (Figure 2) that was later verified with H&E stained sections acquired from a coronal plane perpendicular to the imaging plane. SRS microscopy was also
validated with fresh human GBM specimens and corresponded with H&E analysis. SRS microscopy cannot provide all the current architectural, genetic, and biochemical data available from tissue sectioning, but potentially permits real-time discrimination of glioma boundaries and normal brain, limited to only 100 mm of spatial resolution. Furthermore, SRS microscopy image quality is affected by respiratory and cardiac cycles. Future advances in applying this technology, such as via intraoperative handheld SRS microscopy, may permit intraoperative rapid detection of residual tumor at resection edges, thereby maximizing safe surgical resection and improving glioma patient outcomes. KELLI B. POINTER RAY R. ZHANG JOHN S. KUO ROBERT J. DEMPSEY
VOLUME 74 | NUMBER 2 | FEBRUARY 2014 | N13
Copyright © Congress of Neurological Surgeons. Unauthorized reproduction of this article is prohibited.
SCIENCE TIMES
Figure 2. In vivo SRS microscopy images of human GBM xenografts. Images are representative of 6 mice. SRS imaging was carried out via acute cranial window preparation in mice 24 days after implantation of human GBM xenografts. A, bright-field microscopy appears grossly normal, whereas SRS microscopy within the same FOV demonstrates distinctions between tumorinfiltrated areas and non-infiltrated brain (normal), with a normal brain–tumor interface (dashed line). B-D, high-magnification views within the tumor (B), at the tumor-brain interface (C), and within normal brain (D). (From [Ji M, Orringer DA, Freudiger CW, et al. Rapid, label-free detection of brain tumors with stimulated Raman scattering microscopy. Sci Transl Med. 2013;5 (201):201ra119]. Reprinted with permission from AAAS).
REFERENCE 1. Ji M, Orringer DA, Freudiger CW, et al. Rapid, label-free detection of brain tumors with stimulated Raman scattering microscopy. Sci Transl Med. 2013;5 (201):201ra119.
Knockdown of LGR5 Suppresses the Proliferation of Glioma Cells in Vitro and in Vivo
G
liomas are the most common primary brain tumor in adults and are categorized into 1 of 4 WHO grades based on histopathological characteristics.1 The aggressive, infiltrative nature of high-grade gliomas portends a grave prognosis despite aggressive chemotherapy, radiation, and surgical resection. Current efforts for developing novel and effective treatment options for gliomas have focused on molecular targets that interrupt tumor proliferation. However, these techniques require a thorough understanding of the cell signaling mechanisms of gliomas. Previous research has
N14 | VOLUME 74 | NUMBER 2 | FEBRUARY 2014
delineated a number of proteins critical for normal growth and development that includes regulation of ligand-mediated signaling, cell adhesion, and formation of the extracellular matrix for cell migration.2,3 The leucine-rich repeat containing G protein-coupled receptor 5 (LGR5), has been recently shown to play a key functional role in normal cellular development.4 When abnormal expression of LGR5 is present, tumorigenesis has been shown to occur through activation of the Wnt pathway. However, the exact mechanisms of LGR5 activity in glioma cells have not been elucidated. Recent research published in the October edition of Oncology Reports from Zhang et al in China have confirmed the expression of LGR5 in human gliomas and its correlation with pathological grade and proliferation.5 Additionally, they demonstrate compelling evidence of tumor growth inhibition using RNA interference against LGR5 in both in vitro and in vivo models. The investigators collected 54 gliomas of varying grade from patients who presented for initial surgical resection and had no prior treatments with radiation or chemotherapy. All of the specimens were subjected to immunohistochemical assays for LGR5. LGR5 immunoreactivity
scores (IRS), LGR5 proliferative indices (PI), and the percentage of Ki-67 positive cells were quantified. Both the LGR5 IRS and PI correlated with increasing WHO grade. The investigators then studied the LGR5 expression levels in 3 glioma cell lines (U118, U87, and U251) in vitro and compared them to LGR5 levels expressed in normal human astrocytes. The results demonstrated higher LGR5 expression in the glioma cell lines compared to the normal astrocytes. The team then investigated the role of LGR5 in the malignant transformation of gliomas. The U87 cell lines were transfected with RNAi against LGR5, which resulted in a significant and specific decrease in the endogenous expression of LGR5. In order to evaluate the effect of LGR5 on the growth of U87 cells, the researchers conducted viability assays using U87, U87negative control (NC), and U87-knockdown (KD) cells. Their data showed a marked growth inhibition of U87-KD cells compared to the other groups. Additionally, there were no significant differences in cell growth between the U87 and U87-NC cells. Knockdown of LGR5 also decreased the number and size of colony formation in U87-KD cells
www.neurosurgery-online.com
Copyright © Congress of Neurological Surgeons. Unauthorized reproduction of this article is prohibited.
