[Multimodal method of tissue differentiation in neurooncology using Raman spectroscopy, fluorescence and diffuse reflectance spectroscopy]. / Mul'timodal'nyi metod differentsiatsii tkanei v neiroonkologii s ispol'zovaniem spektroskopii kombinatsionnogo rasseyaniya, fluorestsentsii i diffuznogo otrazheniya.

BACKGROUND: There is a need to expand the possibilities of urgent analysis of intracranial tumor type during resection. These measures are necessary to improve resection quality with preservation of intact tissues and avoiding recurrence and neurological impairment in postoperative period. OBJECTIVE: To create optical-spectral method for differentiating the intracranial tumor types. MATERIAL AND METHODS: We used a combination of certain methods such as fluorescence spectroscopy to analyze the content of endogenous and exogenous fluorophores in samples, diffuse reflectance spectroscopy to analyze structural integrity of tissues according to light scattering and blood filling according to hemoglobin spectrum absorption, as well as spontaneous Raman spectroscopy detecting individual molecular components of tissues. The study was conducted at the Laboratory of Neurosurgical Anatomy and Conservation of Biological Materials of the Burdenko Neurosurgical Center and included 93 tissue samples from 60 patients diagnosed with glioblastoma (n=28), meningioma (n=12), astrocytoma (n=9), oligodendroglioma (n=5), and metastasis (n=6). RESULTS: Different types of intracranial tumors that cannot be differentiated using one of the considered spectroscopy modes can be distinguished in another one. Thus, we can conclude possible advantages of combined optical-spectral approach.

[Intraoperative vascular fluorescence in cerebral glioblastomas and vascular histological features]. / Intraoperatsionnaia fluorestsentsiia sosudov v strukture glioblastom golovnogo mozga i ikh gistologicheskaia kharakteristika.

5-ALA intraoperative fluorescence is widely used in surgery of brain tumors for intraoperative demarcation of boundaries and more total resection because 5-ALA metabolites are not accumulated in the intact brain and vascular tissues. Given this fact, it was hypothesized that fluorescence of vessels in the immediate vicinity of a brain tumor may indicate their infiltration by tumor cells as a potential pathway for their dissemination and as a factor for continued tumor growth after surgery and adjuvant therapy. PURPOSE: Identification of fluorescent vessels located near cerebral gliomas, with a histological description of their structure, relationships with the tumor, and potential invasion of the walls by tumor cells. MATERIAL AND METHODS: A prospective cohort study included 14 patients with malignant supratentorial gliomas, aged 20 to 78 years. Five patients were operated on due to continued tumor growth. Two hours before surgery, all patients received 5-ALA orally. During surgery, a microscope (Carl Zeiss OPMI Pentero, Germany) with a fluorescent module (BLUE-400) was used. In all cases, molecular-genetic and immunohistochemical examinations of the tumor material were performed. During surgery, fluorescent vessels, after evaluating their functional significance, were also resected for histological examination. RESULTS: Glioblastoma and anaplastic astrocytoma were verified in 10 and 4 patients, respectively. In 4 out of 10 glioblastoma cases, vessels with homogeneous or fragmentary fluorescent walls were detected in the tumor bed after resection of most of the tumor; in patients with anaplastic astrocytomas, vascular fluorescence was not observed. In the four vascular samples with intraoperatively detected wall fluorescence, tumor invasion into the vascular layers was revealed in all cases. These patients underwent an immunohistochemical examination with monoclonal antibodies to the glial GFAP marker, which clearly identified areas of ingrowth of tumor cells into the vascular wall. CONCLUSION: 5-ALA intraoperative fluorescence is a fundamentally new approach in the rapid diagnosis of tumor-infiltrated blood vessels. Invasion of tumor cells to intact vessels may be a mechanism of tumor progression and dissemination. Additional resection of fluorescent vessels may affect the radicalness of surgical treatment, but requires a mandatory assessment of their functional significance.

[Intraoperative fluorescence diagnostics in surgery of intracranial meningiomas: analysis of 101 cases]. / Intraoperatsionnaia fluorestsentnaia diagnostika v khirurgii intrakranial'nykh meningiom: analiz 101 nabliudeniia.

Fluorescence diagnostics has been extensively applied in surgery of malignant brain gliomas. However, the use of this technique in surgery of intracranial meningiomas has remained controversial. OBJECTIVE: The study objective was to assess the sensitivity of 5-aminolevulinic acid-based (5-ALA) fluorescence diagnostics in surgery of brain meningiomas and to clarify the clinical and biological factors that may influence the fluorescent effect. MATERIAL AND METHODS: The study consistently included 101 patients with intracranial meningiomas of various locations who were operated on using 5-ALA. There were 28 (27.72%) males and 73 (72.27%) females (median age, 54 years). In all patients, surgery was performed using an operating microscope equipped with a fluorescent module; in 24 of these, laser spectroscopy was used. For comparison of chances to observe the fluorescent effect of 5-ALA in patients having meningiomas with different WHO histological grades (Grade I vs Grade II-III), we performed a meta-analysis that included 10 studies (the largest series) on outcomes of surgical treatment of meningiomas using intraoperative fluorescence diagnostics. RESULTS: Of 101 patients included in this series, observable fluorescence was detected in 95 (94.1%) patients: weak fluorescence in 12 (11.9%), moderate fluorescence in 23 (22.8%) cases, and strong fluorescence in 60 (59.4%) patients. There was no statistically significant relationship (p>0.05) between the rate and intensity of observable fluorescence and the tumor growth pattern (primary/continued), location, WHO grade of malignancy, and histological subtype. In the absence of intraoperative bleeding, tumor fluorescence was statistically significantly brighter (p=0.02). Of 26 patients with hyperostosis, bone fluorescence was observed in 11 (42.3%) cases. There was no statistically significant relationship between administration of dexamethasone, its dose, administration of anticonvulsants, gastrointestinal tract diseases, as well as diabetes mellitus and the fluorescence intensity. There was also no significant relationship between the extent of tumor resection (Simpson scale) and the presence of fluorescence as well as its intensity. Comparison of the observable fluorescence intensity and the laser spectroscopy indicators revealed a significant correlation (r=0.75; p=0.005). CONCLUSION: Meningioma is a well fluorescent tumor, with the technique sensitivity being 94.1%. In some cases, the use of fluorescence diagnostics in surgery of meningiomas improves identification of residual tumor fragments and enables correction of a surgical approach. To assess the effect of fluorescence diagnostics on the recurrence rate and disease-free duration, further research is required.

[Intraoperative fluorescence diagnostics upon recurrent operations for brain gliomas].

UNLABELLED: The method of fluorescent diagnosis (FD) using 5-ALA has been widely employed in surgery of primary intracerebral tumors over the last years. The issue of FD application in surgery of gliomas with continued growth has remained less studied. OBJECTIVE: To investigate the efficacy of using FD with 5-ALA and laser spectral analysis in surgery of brain gliomas with continued growth. MATERIAL AND METHODS: 19 patients with Grade II-IV (WHO) gliomas with continued growth of different localization were studied. All tumors localized supratentorially. 5-Aminolevulinic acid hydrochloride "Alasens" (SSC "NIOPIK", Moscow, Russia) was used in the study. The equipment used during surgery included an operating microscope with an attachment for fluorescent navigation. Apart from expert qualitative assessment of a fluorescence extent, computer analysis of the fluorescence and light scattering (diffuse reflection) spectra was performed on a LESA-01-BIOSPEK spectrum analyzer (Russia). RESULTS: Detectable fluorescence was obtained in all the cases. PP IX fluorescence indices from 9.05 to 53.97 (the study was conducted in 12 cases) were determined by quantitative analysis of the spectrograms. The analysis of light scattering revealed its inverse relationship with respect to the fluorescence index. High sensitivity of the method in surgery of gliomas with continued growth requires clarification of method specificity because non-specific accumulation of PP IX in the area of post-radiation necrosis may occur in these patients. CONCLUSIONS: The FD method can be used for intraoperative demarcation of tumor resection boundaries in surgery of cerebral gliomas with continued growth. However, it is necessary to be critical of the high sensitivity of the method in patients with postradiation pathomorphism due to possible non-specific accumulation of PP IX in tissues. A light scattering study may provide additional information about the structure of tissues in the surgical wound.

[Obtainment of chimeric blastocysts of mice by methods of laser nanosurgery].

The procedure of obtainment of chimeric blastocysts of mice by laser nanosurgery methods without using any other techniques is described. To perform the experiments, a special laser micromanipulator was invented. The murine embryonic stem cells (ESC), which were transformed with pEF-GFP vector, encoding the green fluorescent protein, were used in the experiments. ESC were introduced into the perivitelline space of murine embryos at the stage of 8 cells using the laser micromanipulator. The operated embryos were cultured in vitro until the stage of emergence from zona pellucida. The fluorescence and its precise localization were registered using a confocal microscope. It was shown for the first time that the inclusions of ESC introduced with the lased micromanipulator were found not only in the inner cell mass (ICM) but also in the trophectoderm of the chimeric blastocyst. The technology of nanosurgical operations at early stage preimplanted mammalian embryos using laser techniques opens great opportunities not only for solution of fundamental tasks of experimental embryology of mammals but also for obtainment of chimeric and transgenic animals with predetermined genotype.

[Intraoperative combined spectroscopy (optical biopsy) of cerebral gliomas]. / Intraoperatsionnaia kombinirovannaia spektroskopiia (opti-cheskaia biopsiia) gliom golovnogo mozga.

Clinical studies have revealed high selectivity of 5-ALA-induced protoporphyrin IX accumulation in different brain tumors. Modern methods of evaluation of tissues visible fluorescence are based on the qualitative analysis of the images. Up-to-date methods of combined spectral analysis allow fulfilling the intraoperative quantitative evaluation of the protoporphyrin IX content, as well as the scattering and absorption properties of a tissue. This paper presents a new method of the simultaneous analysis of hemoglobin concentration in oxygenated and reduced forms, tumor marker concentration (5-ALA-induced PP IX) and a new way to analyze the changes in the scattering properties of the tissues. The method is implemented by splitting the visible spectrum into intervals where hemoglobin and protoporphyrin IX have the characteristic peaks of absorption and fluorescence. The present method shows the dependence of the fluorescence index from the tumor grade. Combined spectroscopy (optical biopsy) can detect the differences between the subtypes of gliomas that are similar in the protoporphyrin IX fluorescence index. This method complements and enhances the diagnostic capabilities of spectroscopy, which is particularly important in the non-fluorescent glioma surgery.

[Intraoperative fluorescent visualization and laser spectrosopy in intrinsic brain tumor surgery].

Absence of well-defined borders of the glial tumor due to their infiltrative growth is one of the main issues in neurosurgery. A number of methods for intraoperative visualization are available today. The fluorescent metabolic navigation with 5-aminolevulinic acid (5-ALA) combined with quantitative laser spectroscopy is one of the latest technique. In our series of 99 consecutive patients with brain gliomas (WHO Grade I-IV) we found that visible fluorescence was observed in 68% of cases. Additional use of the laser spectroscopy could increase method sensitivity up to 74% due to accumulation of the protoporphyrine IX in nonfluorescense tumors. It was shown that there are some differences in quantitative fluorescence not only within same tumor (glioblastoma) but also in-between low- and high-grade gliomas. Intraoperative fluorescence and laser spectroscopy are effective and very helpful methods of intraoperative imaging in of intrinsic brain tumor surgery.

[First experience of photodynamic diagnosis and laser spectroalanysis in surgery of intracranial meningiomas].

Photodynamic diagnosis (PDD) has been actively implemented into neurooncological practice, especially in cerebral gliomas surgery. This paper describes our first experience of PDD combined with laser spectroanalysis in intracranial meningiomas. The study included 21 patients (8 male and 13 female patients, mean age was 58 years, range--between 37 and 74 years) with intracranial meningiomas operated with PDD in Burdenko Neurosurgical Institute between 2008 and 2011. In 14 cases laser spectroanalysis was used. Tumor fluorescence was present in all but one cases (95%). Spectroanalysis demonstrated that peaks of fluorescence varied between 5 and 46 (mean level was 18.5). These data correlated with visual impression of fluorescence and confirmed that meningioma is a tumor with bright fluorescence. Radical removal (Simpson grade I-II) was achieved in 10 cases, subtotal resection was performed in the rest of the patients. Application of PDD and laser spectroanalysis allows gaining complete information about accumulation of photosensibilizer in the tissue. To our opinion, these methods may be the most useful for determination of the borders of dural and bony invasion which directly affects the surgical tactics and degree of radical removal. Further studies are needed to evaluate the influence of PDD and laser spectroanalysis on long-term surgical outcomes.

[Photodynamic diagnosis and laser biospectroscopy as an intraoperative guidance in neurosurgery].

Different techniques are used today in neurosurgery for intraoperative navigation, including metabolic guidance using 5-aminolevulinic acid (5-ALA). The article focuses on history of intraoperative photodynamic diagnosis (PDD), mechanisms of 5-ALA action, possibilities of its application in different areas of neurosurgery. In addition to visual assessment of fluorescence, laser biospectroscopy significantly increases the diagnostic value of PDD. Laser biospectroanalysis is described in details, wide perspectives of its application in neurosurgery are demonstrated.

[Experimental rationale for a transscleral approach to thermotherapy for intraocular tumors, by using a photosensitizer].

The authors present the results of an experimental study of the pattern of distribution of laser radiation at a wavelength of 810 nm in the normal and Photosens-photosensitized eye tissues and analyze the distribution of a temperature at the sclera/choroidea/ tumor border during transscleral radiation delivery. To further the goal, the authors have resorted to computer mathematical simulation. In convergent-beam radiation supply, the proportion of a scattered radiation component has been ascertained to be lower in the surface scleral layer than that in the layers adjacent to the choroidea and the base of a tumor. On entering the sclera, a splash of light decreases and power density and radiation-induced heating of the deeply located tissues increase. Thus, the major hyperthermic effects show themselves in the tumor rather than on the sclera.

[Feasibilities of photodynamic therapy in the treatment of corneal mycotic lesions: an experimental study].

The paper presents the results of studying the photofungicidal activity of a photosens (octacationic phthalocyanine) in the in vitro and in vivo systems. Cultured Candida guilliermondii were employed in in vitro investigations. The single combined action of octacationic phthalocyanine at a concentration of up to 1 microM and laser irradiation at a wavelength of 675 nm were established to allow the high activation (up to 100%) of yeast fungi to be achieved in the in vitro system. In vivo investigations were performed on 14 rabbits (28 eyes) with induced keratomycosis. In the first series, fluorescent diagnosis of photosens accumulation in the pathological corneal areas was made in 9 (18 eyes). Based on the data obtained in the second series of experiments, the authors examined the feasibilities of photodynamic therapy for corneal mycotic lesions with the above photosensitizer in 5 rabbits (10 eyes).

[Photodynamic therapy in treatment of chorioid neovascularization].

The authors studied the effectiveness of photodynamic therapy (PDT) with Photosence, a Russian photosensitizer, in treatment of chorioid neovascularization (CNV) in cases of age-related macular degeneration (ARMD) and pathological myopia (PM). The subjects were 73 patients with CNV suffering from ARMD and PM. The efficiency of PDT and complex conservative therapy was compared using vision acuity measurement, retinal morphometry, and fluorescent eye ground angiography (FEGA), performed before treatment, immediately after treatment, and 1, 3, 6, and 12 months later. The study showed that PDT in patients with CNV, ARMD and PM was more efficient than pharmacotherapy. Vision acuity improved or stabilized, and the parameters of retinal morphometry and FEGA improved as well. The results of the study evidence high efficiency of PDT with Photosence in treatment of CNV with ARMD and PM.

[Photodynamic therapy for subretinal neovascular membranes. Communication 2. Results of treatment for complicated myopia].

The efficiency of photodynamic therapy (PDT) versus drug therapy was evaluated in patients with subretinal neovascular membranes (SNM) in complicated myopia (CM). Photosens (aluminum phthalocyanine) was intravenously injected in a dose of 0.05 mg/kg. The irradiation conditions were as follows: a session was carried out, using a laser at a wavelength of 675 nm, in an exposure light dose of 120 J/cm2. The number of sessions ranged from 3 to 5 a week, depending on the clinical picture of SNM. The total light dose was not greater than 500 J/cm2. Twelve months after drug therapy and a course of PDT, reduced visual acuity was observed in 50% and only 20.8% of cases, respectively. Stabilization or increase of visual functions occurred in the remaining patients receiving a course of PDT.

[Role of fluorescence diagnosis using Photosens in patients with subretinal neovascular membrane].

The paper shows the capacity of fluorescence diagnosis (FD) in patients with subretinal neovascular membranes. Photosens was used as a photosensitizer (PS) (NIOPIK) in FD. The agent was intravenously injected in a dose of 0.05 mg/kg. To study the kinetics of tissue PS distribution, the authors employed a procedure of local fluorescence spectroscopy using a LESA-01-Biospek spectrometer, which was improved when applying the procedure for visual assessment of a visual fluorescence image. It was ascertained that the results of photodynamic therapy (PDT) should be compulsorily taken into account in processing its parameters. In some cases, PDT may be recommended on days 3, 5, and 7 after administration of an agent. By taking into account the pharmacodynamics of Photosens, 0.5 mg/kg, all patients should keep the light regime for 3 months.

[Photodynamic therapy for subretinal neovascular membranes. Communication 1. Results of treatment for age-related macular degeneration].

The purpose of the study was to assess the results of photodynamic therapy (PDT) for subretinal neovascular membranes (SNM) in age-related macular degeneration (ARMD), by using the Russian drug Photosens. According to the treatment performed, all the patients were divided into 2 groups: 1) 18 patients with the neovascular form of ARMD who received a course of PDT; 2) 14 patients with the same form who had drug therapy. Photosens (aluminum phthalocyanine) was intravenously injected in a dose of 0.05 mg/kg. The irradiation conditions were as follows: a session was carried out, using a laser at a wavelength of 675 nm, in an exposure light dose of 120 J/cm2. The number of sessions ranged from 3 to 5 a week, depending on the clinical picture of SNM. The total light dose was not greater than 500 J/ cm2. PDT showed a higher efficiency, as compared to drug therapy. PDT using Photosens increases and stabilizes visual acuity in 50% of cases, improves retinal functional activity (an increase in the mean value of a b-wave amplitude), and causes positive changes in the morphometric values of the mean neuroepithelial thickness above SNM and in the foveola.

[Experimental assessment of the capacities of use of photosense. Communication 1. Fluorescence diagnosis of epibulbar and choroid tumors].

Whether Photosense (PS) may be used in the fluorescence diagnosis (FD) of experimental epibulbar and choroid tumors is discussed. The fluorescent ability of Photosense in FD permits detection of advanced malignancies. The kinetics of concentration of PS and the selectivity of its accumulation in malignant versus normal tissues were investigated. PS is shown to be an effective photosensitizer in the FD and, possibly, photodynamic therapy of tumors.

[The first results of phase IIA of clinical studies of photodynamic therapy for subretinal neovascular membranes with photosense].

The subretinal neovascular membrane (SNM) is a common complication of diseases, such as senile macular degeneration, myopia, pseudohistoplasmic syndrome, and many others. There is no algorithm of treatment for this pathology in today's ophthalmological care; however, many authors have arrived at the conclusion that photodynamic therapy (PDT) plays a major role in solving this difficult problem. Fifteen (18 eyes) patients, including 8 (8 eyes) patients diagnosed as having senile macular degeneration and SNM and 7 (10 eyes) patients diagnosed as having complicated high-grade myopia, and 3 were diagnosed as having bilateral SNM. The Russian agent Photosense, 0.05 mg/kg, was used as a photosensitizer. PDT was performed on day 3 after administration of the agent according to the peak of accumulation and the maximum laser concentration gradient at a wavelength of 675 nm and a light dose of 120 J/cm2. Good functional and angiographic results may be observed after the first 3 months of a prospective controlled open-labeled study of the efficiency of PDT with Photosense; however, longer follow-ups and larger groups of patients are required to make a final assessment of this treatment.

[A device for fluorescence diagnosis and photodynamic therapy of eye diseases, by using photosense].

By having a high photodynamic effectiveness and an ability of fluorescence, a Photosense photosensibilizer provides a way of combining photodynamic therapy (PDT) and monitoring its control within a session, which enhances the efficiency of treatment for the subretinal neovascular membrane. A slit lamp-based apparatus complex has been developed to employ the methods of fluorescence diagnosis (FD) and PDT, by applying this photosensitizer. The complex comprises an optical adapter that focusing laser radiation on the fundus of the eye in a range of 100-1000 microm, a video adapter that includes color and high-sensitive monochromic video cameras, as well as a personal computer and software that processes video information from the high-sensitive camera and displays the obtained images in real time. The original system of filters provides an image of the eye fundus in the fluorescent and usual color light at once during a FR procedure. The spatial resolution of the developed apparatus was tested on the test object specially devised for these purposes, which was 10 microm. The sensitivity of the complex is sufficient to record slightly fluorescent objects on the fundus of the eye.

[Experimental assessment of the capacities of use of photosense. Communication 2. Photodynamic therapy for epibulbar and choroid tumors].

The study was undertaken to evaluate the efficiency of photodynamic therapy of pigmented choroid and epibulbar melanoma, by using the agent Photosense. Pigmented choroid and epibulbar melanoma were identified in 50 rabbit eyes. After intravenous injection of Photosense, 0.7 mg/kg, the tumors were irradiated at 675 nm with an argon-pumped dye laser at 150 J/cm2. Photodynamic therapy with Photosense may be effective in treating pigmented choroid and epibulbar melanomas.

[Study of optical absorption of sensitizers in vivo].

The subject of the paper is study of optical absorption of sensitizers in biological tissue. The study shows that absorbance can be used as a tool that allows studying biodistribution of sensitizers and their interaction with tissue in vivo. The article presents a simple technique of determining biological tissue absorption in vivo, and discusses the results of experimental animal studies of some sensitizers.