A method for establishing a tumor-bearing animal model for an OCT system

Abstract

The application belongs to the technical field of animal model construction, and discloses a method for establishing a tumor-bearing animal model for an OCT system, wherein a SD rat supratentorial brain parenchymal glioma model, a SD rat chiasma glioma model and a SD rat brain stem glioma model are constructed by using a C6 cell line; a rat sciatic nerve sheath tumor model and a rat trigeminal nerve RT4 tumor model are constructed by using an RT4 cell line to obtain a SD rat RT4 tumor cell animal model; and a BALB / c nude mouse U87MG tumor cell animal model and a BALB / c nude mouse IOMM-Lee tumor cell animal model are constructed by using a U87MG cell line and an IOMM-Lee cell line, respectively. The application successfully constructs a rat brain glioma model, a chiasma glioma model, a trigeminal nerve sheath tumor model, a sciatic nerve sheath tumor model, a nude mouse brain glioma model and a nude mouse sciatic nerve meningioma model.

Description

Technical Field

[0001] The present invention belongs to the technical field of animal model construction, and in particular relates to a method for establishing a tumor-bearing animal model for an OCT system. Background Art

[0002] Currently, surgical treatment of intracranial tumors has entered the era of precision medicine, requiring neurosurgeons to achieve the most complete resection possible for tumor tissue while effectively preserving normal brain tissue to achieve lower tumor recurrence rates and more complete postoperative neurological function. For gliomas, for example, extended resection of nonfunctional areas is currently widely used in clinical practice to achieve the most complete resection possible. However, if the tumor is located in critical functional areas such as the brainstem, basal ganglia, motor areas, and language areas, surgeons cannot achieve complete resection through extended resection. Therefore, to maximize tumor resection, minimize residual tumors, and effectively identify tumor margins, protect vital neurological functions, and improve progression-free survival, OCT devices offer noninvasive, high-resolution capabilities and have the potential to identify both invasive intracranial tumors and peritumoral normal brain tissue. However, accurate identification of specific tissue types as tumor or normal requires removal of the tissue and gold-standard pathological examination. This approach is invasive and cannot verify tumor margins in critical functional areas (such as the brainstem and basal ganglia) or around the nerves. This has prevented effective verification of the reliability of OCT devices for the diagnosis of infiltrative tumors (such as gliomas) and adhesive tumors (such as schwannomas) in critical brain regions. Establishing in vivo animal models of infiltrative gliomas in critical brain regions and neural adhesive tumors is crucial for validating the effectiveness, practicality, and scientific validity of OCT systems.

[0003] In vitro cell models are suitable for high-throughput drug screening, but the in vitro culture conditions for tumors differ significantly from those in vivo. They lack the in vivo interactions with heterogeneous cells such as neurons, astrocytes, and microglia in the tumor microenvironment. Consequently, screened drugs often fail to validate their efficacy in in vivo animal models. Furthermore, organ culture models for tumor-bearing brain tissue are still relatively scarce.

[0004] Through the above analysis, the problems and defects of the existing technology are as follows:

[0005] 1) There is a lack of reports on animal models of intracranial gliomas located in important functional areas (such as the brainstem and basal ganglia), as well as animal models in which adhesively growing tumors originate from the sciatic nerve.

[0006] 2) The establishment of an animal model of invasive tumor growth is crucial for the pathological characteristics of unclear boundaries between tumor and brain tissue. This technology is crucial for use in OCT systems, enabling the system to detect and identify the boundaries between tumor and brain tissue, and is crucial for more thorough tumor removal and better protection of important brain functional areas.

[0007] 3) The animal model of adhesive tumor growth is useful for verifying whether the OCT system can provide prompts and warnings for nerves located deep within the tumor, thereby reminding the surgeon to operate gently, which plays a positive role in better nerve protection.

[0008] 4) Currently, in vitro cell models are suitable for high-throughput drug screening. However, the in vitro culture conditions for tumor-bearing cells differ significantly from those in vivo. These models lack the in vivo interactions with heterogeneous cells such as neurons, astrocytes, and microglia in the tumor microenvironment. Consequently, screened drugs often fail to validate their efficacy in in vivo animal models. Furthermore, organ culture models for tumor-bearing brain tissue are still relatively scarce.

[0009] To address these issues and drawbacks, further research and exploration are needed to establish tumor-bearing animal models more suitable for OCT systems. This requires interdisciplinary collaborative research involving experts in neurosurgery, pathology, cell biology, biomedical engineering, and pharmacology. Summary of the Invention

[0010] In response to the problems existing in the prior art, the present invention provides a method for establishing a tumor-bearing animal model for an OCT system.

[0011] The present invention is achieved by providing a method for establishing a tumor-bearing animal model for an OCT system, the method comprising:

[0012] The C6 cell line was used to establish tumor-functional brain parenchyma / brain nerve infiltration models in SD rats, including SD rat brainstem glioma models, basal ganglia glioma models, and optic chiasm glioma models;

[0013] SD rat tumor-nerve adhesion models were established using the RT4 cell line, including the sciatic nerve RT4 tumor model and the trigeminal nerve RT4 tumor model;

[0014] The U87MG cell line was used to construct a BALB / c nude mouse tumor-brain parenchyma infiltration growth model, including a BALB / c nude mouse brainstem glioma model and a basal ganglia glioma model; and the IOMM-Lee cell line was used to construct a BALB / c nude mouse tumor-sciatic nerve adhesion model, namely the IOMM-Lee tumor cell animal model.

[0015] Furthermore, the method for establishing a tumor-bearing animal model for the OCT system includes the following steps:

[0016] Step 1: C6 animal models were constructed by culturing C6 cell lines and preparing C6 cells to obtain supratentorial parenchymal glioma models, chiasmatic glioma models, and brainstem glioma models in SD rats.

[0017] Step 2: Developing a SD rat RT4 tumor cell animal model by culturing the RT4 cell line, preparing RT4 cells, and constructing an RT4 animal model to construct a rat sciatic nerve sheath tumor model and a rat trigeminal nerve RT4 tumor model;

[0018] Step 3: Cultivate the U87MG cell line, prepare U87MG cells, and establish a BALB / c nude mouse U87MG tumor cell animal model;

[0019] Step 4: Culture the IOMM-Lee cell line, prepare IOMM-Lee cells, and establish a BALB / c nude mouse IOMM-Lee tumor cell animal model.

[0020] Furthermore, the method of culturing a C6 cell line, preparing C6 cells, and constructing a C6 animal model, a SD rat supratentorial brain parenchymal glioma model, a SD rat optic chiasm glioma model, and a SD rat brainstem glioma model comprises:

[0021] (1) Obtain C6 cells and culture them in a 37°C, 5% CO2 incubator for 24 hours. Change the medium according to the cell growth status and the color change of the culture medium. When the cells have grown to 80%-90% of the bottom area of ​​the bottle, subculture the C6 cells at a ratio of 1:2 to 1:3. Continue to culture the subcultured C6 cells in a 37°C, 5% CO2 incubator using high-glucose DMEM medium containing 10% fetal bovine serum. Change the culture medium every 1-2 days according to the growth status, and observe and record the cell growth status every day.

[0022] (2) After the C6 cells reached the passage conditions, they were rinsed with PBS three times, and 1 mL of trypsin was added and incubated at 37°C for 1 min. The digested cells were rinsed with complete medium and centrifuged at 800 rpm for 3 min to remove the trypsin, FBS, DMEM and other waste liquids. 2 mL of PBS was added and pipetted evenly, and then centrifuged at 800 rpm for 3 min and repeated three times. After centrifugation, the supernatant was discarded and an appropriate amount of PBS was added to dilute the cell density to 10 4 / uL; aspirate 10uL of cell suspension for cell counting, and calculate the total number of cells in the centrifuge tube based on the cell count value; aspirate PBS containing a small amount of cell debris and residual DMEM, and then add 5mL PBS to prepare a cell suspension;

[0023] (3) Digest and resuspend C6 cells in the logarithmic growth phase in 5 mL of PBS, take 10 μL to count the cells, and adjust the viable cell concentration to 1×10 4 / uL, 5×10 4 / uL, 5×10 4 The supratentorial parenchymal glioma model, the chiasmatic glioma model and the brainstem glioma model of SD rats were established respectively.

[0024] Furthermore, the C6 cell passaging includes: preheating 0.25% trypsin-0.53mM EDTA digestion solution in a 37°C water bath, removing the culture medium in the culture flask, adding 4mL of PBS to the culture flask, gently shaking and washing, and then discarding; adding 2ml of preheated trypsin to the flask, incubating at 37°C for digestion for 1 minute, and adding 2ml of complete culture medium to terminate digestion after digestion; gently blowing the cells on the flask wall with a pipette, collecting the cell suspension after the cells on the flask wall are completely detached, centrifuging at 800rpm for 3min, discarding the supernatant, adding complete culture medium to resuspend the cells, and transferring the cells to a 10cm culture dish for passaging.

[0025] Furthermore, the SD rat RT4 tumor cell animal model is obtained by culturing the RT4 cell line, preparing the RT4 cells, and constructing the RT4 animal model to construct a rat sciatic nerve sheath tumor model and a rat trigeminal nerve RT4 tumor model, including:

[0026] 1) Cultivation of RT4 cell line: RT4 cells were cultured in a 37°C, 5% CO2 incubator for 24 hours. The medium was changed based on cell growth and culture medium color change. When cells covered 80%-90% of the flask bottom area, the RT4 cells were passaged at a ratio of 1:2 to 1:3. The passaged RT4 cells were then cultured in high-glucose DMEM medium supplemented with 10% fetal bovine serum in a 37°C, 5% CO2 incubator. The culture medium was changed every 1-2 days based on cell growth, and cell growth was observed and recorded daily.

[0027] 2) Preparation of RT4 cells: After RT4 cells reach passage conditions, rinse three times with PBS, add 1 mL of trypsin, and incubate at 37°C for 3 min. Rinse the digested cells with complete medium and centrifuge at 1000 rpm for 5 min. Remove the supernatant containing trypsin, FBS, DMEM, and other waste liquids. Add 2 mL of PBS and pipette evenly. Centrifuge again at 1000 rpm for 5 min and repeat three times. Discard the supernatant and add PBS to dilute the cells to the desired concentration. Count 10 μL of the cell suspension and calculate the total number of cells in the centrifuge tube based on the cell count. Aspirate the PBS containing a small amount of cell debris and residual DMEM, and add 0.5 mL of PBS to prepare the cell suspension.

[0028] 3) Construction of RT4 animal model: Digest RT4 cells in the logarithmic growth phase and resuspend them in 5 mL PBS. Take 10 μL of cells for cell counting and adjust the viable cell concentration to 1×10 based on the counting results. 4 / uL and 5×10 4 / uL, and established rat sciatic nerve sheath tumor model and rat trigeminal nerve RT4 tumor model.

[0029] Furthermore, the RT4 cell passaging includes: preheating 0.25% trypsin-0.53mM EDTA digestion solution in a 37°C water bath, removing the culture medium in the culture flask, adding 4ml PBS to the culture flask, gently shaking and washing, and then discarding; adding 2ml of preheated trypsin to the flask, incubating at 37°C for digestion for 1 minute, and adding 2ml of complete culture medium to terminate digestion after digestion; gently blowing the cells on the flask wall with a pipette, collecting the cell suspension after the cells on the flask wall are completely detached, centrifuging at 1000rpm for 5 minutes, discarding the supernatant, adding complete culture medium to resuspend the cells, and transferring the cells to a 10cm culture dish for passaging.

[0030] Furthermore, the culturing of U87MG cell lines, preparation of U87MG cells, and establishment of a BALB / c nude mouse U87MG tumor cell animal model include:

[0031] (1) Cultivation of U87MG cell line: Obtain U87MG cells and culture them in a 37°C, 5% CO2 incubator for 24 hours. Change the medium according to the cell growth status and the color change of the culture medium. When the cells have grown to 80%-90% of the bottom area of ​​the bottle, subculture the U87MG cells at a ratio of 1:2 to 1:3. Continue to culture the subcultured U87MG cells in a 37°C, 5% CO2 incubator using high-glucose DMEM medium containing 10% fetal bovine serum. Change the culture medium every 1-2 days according to the growth status, and observe and record the cell growth status every day.

[0032] (2) Preparation of U87MG cells: After U87MG cells reach the passage conditions, rinse the cells three times with PBS, add 1 mL of trypsin and incubate at 37°C for 1 min; rinse the digested cells with complete medium, centrifuge at 800 rpm for 3 min, remove the trypsin, FBS, DMEM and other waste liquids; add 2 mL of PBS, pipette and blow evenly, centrifuge at 800 rpm for 3 min and repeat three times; discard the supernatant after centrifugation, add appropriate amount of PBS to dilute the cell density to 10 4 / uL; aspirate 10uL of cell suspension for cell counting, and calculate the total number of cells in the centrifuge tube based on the cell count value; aspirate PBS containing a small amount of cell debris and residual DMEM, and then add 5mL PBS to prepare a cell suspension;

[0033] (3) Construct the U87MG animal model.

[0034] Furthermore, the U87MG cell passaging includes: preheating 0.25% trypsin-0.53mM EDTA digestion solution in a 37° water bath, aspirating the culture medium in the culture flask, adding 4mL of PBS to the culture flask, gently shaking and washing, and then discarding; adding 2ml of preheated trypsin to the flask, incubating at 37°C for digestion for 1 minute, and adding 2ml of complete culture medium to terminate digestion after digestion; gently blowing the cells on the flask wall with a pipette, collecting the cell suspension after the cells on the flask wall are completely detached, centrifuging at 800rpm for 3 minutes, discarding the supernatant, adding complete culture medium to resuspend the cells, and transferring the cells to a 10cm culture dish for passaging.

[0035] Furthermore, the culturing of the IOMM-Lee cell line, the preparation of IOMM-Lee cells, and the establishment of a BALB / c nude mouse IOMM-Lee tumor cell animal model include:

[0036] 1) IOMM-Lee Cell Line Culture: IOMM-Lee cells were cultured in a 37°C, 5% CO2 incubator for 24 hours. The medium was changed based on cell growth and culture medium color change. When cells covered 80%-90% of the flask bottom area, the IOMM-Lee cells were passaged at a ratio of 1:2 to 1:3. The passaged IOMM-Lee cells were then cultured in high-glucose DMEM medium supplemented with 10% fetal bovine serum in a 37°C, 5% CO2 incubator. The culture medium was changed every 1-2 days based on cell growth, and cell growth was observed and recorded daily.

[0037] The IOMM-Lee cell passaging method includes: preheating a 0.25% trypsin-0.53 mM EDTA digestion solution in a 37°C water bath, removing the culture medium from the culture flask, adding 4 mL of PBS to the culture flask, gently shaking and washing, and then discarding the solution; adding 2 mL of preheated trypsin to the flask, incubating at 37°C for 1 minute, and adding 2 mL of complete culture medium to terminate the digestion after the digestion is complete; gently blowing the cells on the flask wall with a pipette to collect the cell suspension after the cells on the flask wall are completely detached, centrifuging at 800 rpm for 3 minutes, discarding the supernatant, adding complete culture medium to resuspend the cells, and transferring the cells to a 10 cm culture dish for passaging;

[0038] 2) After the IOMM-Lee cells reached the passage conditions, they were rinsed three times with PBS, and 1 mL of trypsin was added and incubated at 37°C for 1 min. The digested cells were rinsed with complete medium and centrifuged at 800 rpm for 3 min to remove the supernatant containing trypsin, FBS, DMEM, and other waste liquids. 2 mL of PBS was added and pipetted evenly. The supernatant was then centrifuged at 800 rpm for 3 min and repeated three times. After centrifugation, the supernatant was discarded and the cell density was diluted to 10 with an appropriate amount of PBS. 4 / uL; aspirate 10uL of cell suspension for cell counting, and calculate the total number of cells in the centrifuge tube based on the cell count value; aspirate PBS containing a small amount of cell debris and residual DMEM, and then add 5mL PBS to prepare a cell suspension;

[0039] 3) Construction of IOMM-Lee animal model: RT4 cells in the logarithmic growth phase were digested and resuspended in 5 mL PBS. 10 μL was taken for cell counting. The viable cell concentration was adjusted to 1×10 based on the counting results. 4 / uL and 5×10 4 / uL, and establish the IOMM-Lee animal model.

[0040] Another object of the present invention is to provide a tumor-bearing animal model for an OCT system constructed using the method for establishing a tumor-bearing animal model for an OCT system.

[0041] In combination with the above technical solutions and the technical problems solved, the advantages and positive effects of the technical solutions to be protected by the present invention are as follows:

[0042] First, the present invention successfully constructed a variety of tumor-bearing animal models (rat brain glioma model, optic chiasmatic glioma model, trigeminal schwannoma model, sciatic schwannoma model, nude mouse brain glioma model, and nude mouse sciatic meningioma model).

[0043] The present invention constructs a variety of tumor-bearing animal models (rat brain glioma model, optic chiasmatic glioma model, trigeminal schwannoma model, sciatic nerve schwannoma model, as well as nude mouse brain glioma model, nude mouse sciatic meningioma model) to verify the effectiveness and practicality of the constructed OCT system in in vivo detection of various tumor boundaries.

[0044] Second, as auxiliary evidence for the inventiveness of the claims of the present invention, it is also reflected in the following important aspects:

[0045] (1) The expected benefits and commercial value of the technical solution of the present invention after transformation are:

[0046] Detecting tumor boundaries using OCT in living animals is crucial. Loss of blood perfusion and volumetric deformation during specimen acquisition can lead to discrepancies between the OCT grayscale values ​​of ex vivo specimens and those of living specimens. Furthermore, surgical interference with the detection area, such as intraoperative bleeding and hematoma formation, scab formation after bipolar coagulation, and the regular pulsation of brain tissue, can affect the OCT system's detection process and the resulting grayscale image.

[0047] (2) The technical solution of the present invention fills the technical gap in the industry at home and abroad:

[0048] The brainstem glioma, optic nerve glioma, sciatic nerve sheath tumor, trigeminal nerve sheath tumor, and peripheral nerve meningioma models established in this invention, along with their technical solutions for detecting tumor boundaries using an OCT system, represent a technological gap in the industry, both domestically and internationally. This is likely due to the difficulty of performing craniotomy and in vivo OCT detection in the deep location of the tumor, further highlighting the significance and value of this invention in systematically applying OCT systems to detect the boundaries of various tumors. However, no relevant literature or patents, both domestically and internationally, have reported on the application of OCT systems to detect the boundaries of brainstem gliomas or gliomas involving the basal ganglia in tumor-bearing animal models.

[0049] (3) Whether the technical solution of the present invention solves the technical problems that people have been eager to solve but have not been able to solve successfully:

[0050] Establishing tumor-bearing animal models allows the effectiveness and practicality of OCT systems to be verified in these models. This is particularly important for detecting the boundaries of brainstem gliomas and, when supratentorial gliomas invade the basal ganglia, for detecting the boundaries between the glioma and the normal peritumoral basal ganglia region. This is for two reasons: First, conducting such studies in humans is difficult and challenging. Even if an OCT system is used intraoperatively to detect brainstem gliomas and areas associated with gliomas invading the basal ganglia, and OCT grayscale images are obtained, it is impossible to obtain tissue specimens from critical functional areas and obtain pathological confirmation that the detected areas are in the brainstem or basal ganglia. Obtaining tissue specimens from critical functional areas can lead to serious consequences such as paralysis, paresthesias, speech dysfunction, coma, or even death. Therefore, obtaining OCT grayscale images of the tumor and the normal peritumoral brainstem and basal ganglia tissue in animal experiments, along with pathological confirmation, is crucial. Second, detecting the boundaries of gliomas in these areas is of paramount clinical significance. Surgical treatment of intracranial tumors should reflect precision surgery. Precision surgery is even more important for brainstem gliomas and gliomas invading the basal ganglia, as they involve the protection of neurological functions in important functional areas. This invention demonstrates that the OCT system can assist in intraoperative identification of tumor boundaries in animal models of brainstem gliomas and gliomas invading the basal ganglia, thereby confirming the OCT system's ability to assist in precision surgical treatment of gliomas in important neurological functional areas.

[0051] (4) Whether the technical solution of the present invention overcomes technical prejudice:

[0052] The technical solution of the present invention proposes constructing an adhesive tumor model in which the OCT system can detect and differentiate between gliomas and the optic nerve, neurilemmomas and the sciatic and trigeminal nerves, and meningiomas and the sciatic nerve. It also provides an early warning of the optic, sciatic, and trigeminal nerves located deep within the tumor (within 2-3 mm), prompting the surgeon to be gentle, reduce nerve traction, and maximize the likelihood of preserving nerve function. We believe that the role of the OCT system in adhesive tumor models is particularly significant. A review of the literature indicates that the application of the OCT system in intracranial adhesive tumor models has not yet attracted the attention, research, or reports of the inventors and researchers. The research solution of the present invention, particularly the OCT system's early warning function for deep peripheral nerves in adhesive tumor models, demonstrates that the OCT system can not only detect tumor boundaries in invasive tumor models, but also has the potential to identify tumor boundaries in adhesive tumor models during surgery. Unlike neuroelectrophysiological monitoring technology used to determine the course of cranial nerves during surgery, its accuracy depends on direct stimulation of the nerves after exposure. For cranial nerves deeply buried on the ventral side of the tumor tissue or wrapped in the tumor, the sensitivity and specificity of electrophysiological detection are greatly reduced. However, the present invention proves that the OCT system can indicate and warn the course of cranial nerves when the cranial nerves are still located deep in the tumor. This suggests that the OCT system is expected to be similar to neuroelectrophysiological monitoring technology, but with the unique advantage of deep-surface warning function, and has the potential to become an alternative technology for real-time monitoring of the course of cranial nerves in the tumor area during surgery. It also suggests that continuing to improve the performance of the OCT system, especially the improvement in detection depth, will play a positive role in enhancing the boundary warning capability of adhesion tumors and improving the application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 This is a flow chart of a method for establishing a tumor-bearing animal model for an OCT system provided in an embodiment of the present invention;

[0054] Figure 2 is a flow chart of C6 cell preparation provided by an embodiment of the present invention;

[0055] Figure 3 This is a flow chart of stereotactic intracranial tumor implantation in SD rats provided by an embodiment of the present invention;

[0056] Figure 4 This is a flow chart of RT4 cell preparation provided by an embodiment of the present invention;

[0057] Figure 5 This is a flow chart for establishing a sciatic nerve sheath tumor model in SD rats under a microscope provided by an embodiment of the present invention;

[0058] Figure 6 This is a flow chart for preparing U87MG cells provided in an embodiment of the present invention;

[0059] Figure 7 This is a schematic diagram of establishing a U87MG animal model provided by an embodiment of the present invention;

[0060] Figure 8 is a flow chart of IOMM-Lee cell preparation provided by an embodiment of the present invention;

[0061] Figure 9 Schematic diagram of establishing the IOMM-Lee animal model provided by an embodiment of the present invention;

[0062] Figure 10 This is a flowchart of tissue embedding and sectioning provided by an embodiment of the present invention;

[0063] Figure 11 This is a HE staining flow chart provided by an embodiment of the present invention;

[0064] Figure 12 This is a preoperative head magnetic resonance T2 image of the SD rat supratentorial C6 glioma animal model provided in an embodiment of the present invention;

[0065] Figure 13 This is an OCT grayscale image of brain tissue covering the tumor surface removed from the SD rat supratentorial C6 glioma animal model provided in an embodiment of the present invention;

[0066] Figure 14 This is a two-dimensional color image of brain tissue covering the tumor surface removed from an SD rat supratentorial C6 glioma animal model provided in an embodiment of the present invention detected by OCT;

[0067] Figure 15 This is an OCT surface grayscale image of the optic chiasm C6 tumor-bearing SD rat animal model provided by an embodiment of the present invention;

[0068] Figure 16 This is an OCT B-scan grayscale image of the optic chiasm C6 tumor-bearing SD rat animal model provided by an embodiment of the present invention;

[0069] Figure 17 This is a two-dimensional color image of the OCT detection of the optic chiasm C6 tumor-bearing SD rat animal model provided by an embodiment of the present invention;

[0070] Figure 18 This is a preoperative plain scan T2 image of a brainstem C6 tumor-bearing SD rat animal model provided by an embodiment of the present invention;

[0071] Figure 19 This is a grayscale image generated by OCT detection of the exposed tumor and brainstem region in the brainstem C6 tumor-bearing SD rat animal model provided by an embodiment of the present invention;

[0072] Figure 20 This is an OCT grayscale image of a resected tumor in a brainstem C6 tumor-bearing SD rat animal model provided by an embodiment of the present invention;

[0073] Figure 21 This is a two-dimensional color image of a resected tumor detected by OCT in a brainstem C6 tumor-bearing SD rat animal model provided by an embodiment of the present invention;

[0074] Figure 22 This is an OCT grayscale image of a tumor covering the surface of the brainstem after resection in a brainstem C6 tumor-bearing SD rat animal model provided by an embodiment of the present invention;

[0075] Figure 23 This is an OCT two-dimensional color image of a tumor covering the surface of the brainstem after resection of a brainstem C6 tumor-bearing SD rat animal model provided by an embodiment of the present invention;

[0076] Figure 24 This is an OCT grayscale image of the tumor cavity detected by resection of a glioma mass located in the medulla oblongata in a brainstem C6 tumor-bearing SD rat animal model provided by an embodiment of the present invention;

[0077] Figure 25 This is a two-dimensional color image of the tumor cavity detected by OCT in the brainstem C6 tumor-bearing SD rat animal model provided by the embodiment of the present invention after the glioma mass located in the medulla oblongata was removed;

[0078] Figure 26 This is a B-scan grayscale image detected by an OCT system after partial tumor resection in a cerebellar C6 tumor-bearing SD rat animal model provided by an embodiment of the present invention;

[0079] Figure 27 This is a two-dimensional color image detected by an OCT system after partial tumor resection in a cerebellar C6 tumor-bearing SD rat animal model provided by an embodiment of the present invention;

[0080] Figure 28 This is a B-scan grayscale image of the tumor boundary detected by an OCT system after resection of a glioma in a cerebellar C6 tumor-bearing SD rat animal model provided by an embodiment of the present invention;

[0081] Figure 29 This is a two-dimensional color image of the tumor boundary detected by an OCT system after resection of a glioma in a cerebellar C6 tumor-bearing SD rat animal model provided by an embodiment of the present invention;

[0082] Figure 30 This is a B-scan image of an RT4 tumor in the sciatic nerve of a SD rat provided in an embodiment of the present invention;

[0083] Figure 31 This is a two-dimensional color image of an RT4 tumor in the sciatic nerve of a SD rat provided in an embodiment of the present invention;

[0084] Figure 32 This is a B-scan grayscale image of the sciatic nerve of an SD rat after tumor resection provided in an embodiment of the present invention;

[0085] Figure 33 This is a two-dimensional color image of the sciatic nerve of an SD rat after tumor resection provided in an embodiment of the present invention;

[0086] Figure 34 This is a grayscale B-scan image of an RT4 tumor on the right sciatic nerve of an SD rat provided in an embodiment of the present invention;

[0087] Figure 35 This is an OCT scan of a distal sciatic nerve tumor and a B-scan grayscale image of a nerve complex provided by an embodiment of the present invention;

[0088] Figure 36 This is a B-scan grayscale image of the sciatic nerve of a SD rat with a tumor removed provided in an embodiment of the present invention;

[0089] Figure 37 This is a two-dimensional color image of the sciatic nerve of a SD rat with a tumor removed provided in an embodiment of the present invention;

[0090] Figure 38 The SD rat sciatic nerve-bearing RT4 tumor provided in the embodiment of the present invention;

[0091] Figure 39 This is a B-scan grayscale image of the tumor and the normal brain tissue boundary around the tumor detected by the OCT system after the U87 nude mouse tumor is exposed on the upper screen provided by the embodiment of the present invention;

[0092] Figure 40 The embodiment of the present invention provides a two-dimensional color image of the tumor and the normal brain tissue boundary around the tumor detected by the OCT system after the tumor was exposed on the U87 nude mouse.

[0093] Figure 41 The embodiment of the present invention provides a method for performing partial resection of a tumor in a U87 nude mouse with a supratentorial tumor, so that part of the tumor remains at the bottom of the tumor cavity, and performing OCT detection of a B-scan grayscale image.

[0094] Figure 42 The embodiment of the present invention provides a method for performing OCT detection of a two-dimensional color image of a U87 nude mouse with a partial tumor resection, with a portion of the tumor remaining at the bottom of the tumor cavity.

[0095] Figure 43 This is a B-scan grayscale image of an OCT scan performed on a U87 nude mouse with residual tumor at the bottom of the tumor cavity removed after supratentorial loading provided by an embodiment of the present invention;

[0096] Figure 44 This is a two-dimensional color image of OCT detection of a residual tumor at the bottom of the tumor cavity of a supratentorial U87 nude mouse after resection of the residual tumor provided by an embodiment of the present invention;

[0097] Figure 45This is a grayscale image of a nude mouse with a 10MM tumor on the right sciatic nerve provided by an embodiment of the present invention;

[0098] Figure 46 This is a two-dimensional color image of a nude mouse with a right sciatic nerve IOMM tumor provided by an embodiment of the present invention. DETAILED DESCRIPTION

[0099] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0100] The method for establishing a tumor-bearing animal model for an OCT (optical coherence tomography) system provided in an embodiment of the present invention comprises the following steps:

[0101] 1) Establishment of functional brain parenchyma / nerve invasion models in SD rats using the C6 cell line: This step involves establishing different types of SD rat brain tumor models, such as brainstem glioma models, basal ganglia glioma models, and chiasmatic glioma models. These models can be used to study the growth and invasion characteristics of brain tumors.

[0102] 2) Establishment of a SD rat tumor-nerve adhesion model using the RT4 cell line: This step involves establishing sciatic nerve RT4 tumor models and trigeminal nerve RT4 tumor models. These models can be used to study the development of peripheral nerve tumors and nerve adhesion phenomena.

[0103] 3) Establishment of a BALB / c nude mouse brain parenchymal tumor-invasion growth model using the U87MG cell line: This step involves establishing BALB / c nude mouse models of brainstem glioma and basal ganglia glioma. These models can be used to study the growth and invasion characteristics of brain tumors in nude mice.

[0104] 4) The IOMM-Lee cell line was used to establish a BALB / c nude mouse tumor-sciatic nerve adhesion model, also known as the IOMM-Lee tumor cell animal model. This model can be used to study the development of sciatic nerve tumors and nerve adhesion phenomena.

[0105] The working principle and signal processing process of the method for establishing a tumor-bearing animal model for an OCT (optical coherence tomography) system provided in an embodiment of the present invention are as follows:

[0106] OCT systems achieve high-resolution imaging of tissue structures by measuring the reflection and scattering properties of light. OCT uses a low-coherence light source to emit a beam of light, which is split into two paths by an interferometer. One beam illuminates the tissue under examination, while the other illuminates a reference mirror. The two beams are then superimposed at the detector, generating an interference signal. Analysis of this interference signal reveals information about the internal structure of the tissue under examination.

[0107] In this approach, by constructing different types of tumor-bearing animal models, researchers can use OCT systems to perform real-time, non-destructive, high-resolution imaging of these models. This imaging data can help researchers gain a deeper understanding of phenomena such as tumor growth, invasion, and neural adhesion, providing strong support for tumor diagnosis and treatment.

[0108] The signal processing process of the method for establishing a tumor-bearing animal model for an OCT (optical coherence tomography) system provided in an embodiment of the present invention includes the following steps:

[0109] 1) Collect the interference signal generated by the OCT system.

[0110] 2) Preprocess the interference signal, such as denoising and background removal.

[0111] 3) The interference signal is converted from the time domain to the frequency domain through Fourier transform to obtain the depth information of the tissue.

[0112] 4) Quantify and normalize the frequency domain signal based on the optical properties of the tissue, such as reflectivity and scattering coefficient.

[0113] 5) Perform an inverse Fourier transform on the processed signal to convert it from the frequency domain back to the time domain.

[0114] 6) Reconstructing a two-dimensional or three-dimensional image of the tissue structure using the processed signal data.

[0115] 7) Further analysis and processing of the reconstructed images, such as segmentation, feature extraction, and classification, are performed to extract key information about tumor growth, invasion, and neural adhesion.

[0116] This method for establishing tumor-bearing animal models for OCT systems can provide real-time, non-invasive, high-resolution imaging data for studying different types of tumors. This data is crucial for gaining a deeper understanding of tumor growth, invasion, and neural adhesion, and for providing strong support for tumor diagnosis and treatment.

[0117] like Figure 1As shown, the method for establishing a tumor-bearing animal model for an OCT system provided in an embodiment of the present invention includes the following steps:

[0118] S101, by culturing C6 cell lines and preparing C6 cells, a C6 animal model was established to obtain the SD rat supratentorial brain parenchymal glioma model, the SD rat optic chiasm glioma model, and the SD rat brainstem glioma model;

[0119] S102, the SD rat RT4 tumor cell animal model was obtained by culturing the RT4 cell line, preparing RT4 cells, and establishing the RT4 animal model to construct a rat sciatic nerve sheath tumor model and a rat trigeminal nerve RT4 tumor model;

[0120] S103, culture U87MG cell line, prepare U87MG cells, and establish BALB / c nude mouse U87MG tumor cell animal model;

[0121] S104, culture the IOMM-Lee cell line, prepare IOMM-Lee cells, and establish a BALB / c nude mouse IOMM-Lee tumor cell animal model.

[0122] The method for establishing a tumor-bearing animal model for an OCT system provided in an embodiment of the present invention specifically includes:

[0123] 1.1 Establishment of tumor-bearing animal model

[0124] (1) Establishment of the SD rat C6 tumor cell animal model

[0125] (1) Culture of C6 cell line

[0126] Commercial C6 cells were purchased from the Shanghai Cell Bank of the Committee of Typical Culture Collection of the Chinese Academy of Sciences. After thawing, the cells were allowed to adhere to the culture flasks, filled with culture medium, and shipped sterilely. Upon receipt, the cells were cultured in a 37°C, 5% CO2 incubator for 24 hours. The medium was changed and subcultured every two to three days, depending on cell growth and changes in the culture medium color. When cells covered 80%-90% of the flask bottom area as observed under a microscope, they were subcultured at a subculture ratio of 1:2 to 1:3. Subculture procedure: Preheat 0.25% trypsin-0.53mM EDTA digestion solution in a 37°C water bath. Aspirate the culture medium from the culture flask and add 4mL of PBS to the flask. Gently shake and wash, then discard. Add 2mL of preheated trypsin to the flask and incubate at 37°C for 1 minute. After digestion, add 2mL of complete culture medium to terminate the digestion. Use a pipette to gently blow the cells on the wall of the bottle to completely remove them, then collect the cell suspension, centrifuge at 800rpm for 3 minutes, discard the supernatant, add complete culture medium to resuspend the cells, transfer the cells to a 10cm culture dish, and pass them on. After passaging, C6 cells continue to be cultured in a 37°C, 5% CO2 incubator using high-glucose DMEM medium containing 10% fetal bovine serum. Change the culture medium every 1-2 days according to the growth situation, and observe and record the cell growth status, such as status, density, morphology, etc., every day. After the cell growth state stabilizes, it is prepared for SD rat brain glioma and optic chiasm glioma modeling.

[0127] (2) C6 cell preparation

[0128] In addition to the cell washing, EDTA digestion, centrifugation and other processes in the conventional cell culture process, the cell preparation process of the tumor model also includes PBS washing of cells after centrifugation, cell counting and cell concentration adjustment. The trypsin, cell debris, DMEM and other tissues remaining in the tumor cells after centrifugation can be removed by multiple washings with PBS to reduce the local immune or inflammatory response after cell implantation. The specific steps of cell preparation are as follows: Figure 2 shown.

[0129] (3) Establishment of C6 animal model

[0130] Digest and resuspend C6 cells in the logarithmic growth phase in 5 mL PBS, take 10 μL to count the cells, and adjust the viable cell concentration to 1×10 4 / uL, in order to establish the supratentorial brain parenchymal glioma model of SD rats. The injection point is the bregma as the origin (X=0, Y=0), select the point on the skull where X=-3.50mm (negative value represents the right side) and Y=1.00mm, drill through the skull with a Reward drill, and use the pusher to push the 10uL microinjector needle tip to the dura mater at the drill hole, adjust the Z value to zero, enter the skull, and after the Z value reaches 6.00mm, withdraw 1.00mm to inject the tumor suspension. The injection volume is 10uL, and the injection speed is set to 1uL / min. After the injection is completed, leave it for 10 minutes and then slowly withdraw the needle, seal the bone hole with bone wax, suture the scalp, disinfect, and feed normally. The specific operation process and schematic diagram are as follows Figure 3 All animal experiments in this invention were reviewed and approved by the Experimental Animal Ethics Committee of West China Hospital, Sichuan University.

[0131] For the SD rat chiasmatic glioma model, the viable cell concentration needs to be adjusted to 5×10 4 The injection point is the bregma as the origin (X=0, Y=0). After drilling a hole with the bregma, the 10µL microinjector needle tip is positioned against the dura mater at the drilled hole. The Z value is adjusted to zero and the needle is inserted into the brain. When the Z value reaches 9.50mm, needle advancement is stopped and the tumor suspension is injected. The injection volume is 2µL, and the injection rate is set to 0.5µL / min. After the injection is completed, the needle is left in place for 4 minutes before slowly withdrawing.

[0132] The SD rat brainstem glioma model requires the concentration of living cells to be adjusted to 5×10 4 The bregma was used as the origin, and the injection point was set at X = -2.00 mm, Y = -10.56 mm (negative values ​​indicate the caudal direction), and Z = 8.50 mm. The injection volume was 2 μL, and the injection speed was set at 0.5 μL / min. After the injection was completed, the needle was left in place for 4 minutes before slowly withdrawing. The SD rat cerebellar glioma model required the viable cell concentration to be adjusted to 1 × 10 4 The bregma was used as the origin, and the injection point was set at X = -2.00 mm, Y = -11.04 mm, and Z = 4.00 mm. The injection volume was 10 μL, and the injection rate was set at 1 μL / min. After the injection was completed, the needle was left in place for 10 minutes before slowly withdrawing it.

[0133] (II) Establishment of SD rat RT4 tumor cell animal model

[0134] (1) Culture of RT4 cell line

[0135] Commercial RT4 cells were purchased from the ATCC cell bank in the United States. After thawing, the cells were allowed to adhere to the wall of the culture flask and then filled with culture medium and shipped sterilely. The remaining procedures were similar to those for C6 cells. However, due to the rich cytoplasm and larger size of RT4 cells, a higher centrifugal force and longer centrifugation time were required. The centrifugation parameters were set to 1000 rpm for 5 minutes. Once the cells were stable, they were prepared for use in modeling sciatic and trigeminal schwannomas in SD rats.

[0136] (2) RT4 cell preparation

[0137] After centrifugation, the remaining trypsin, cell debris, DMEM and other tissues in the tumor cells can be removed by multiple washings with PBS. Because RT4 cells have more abundant and elongated dendrites and more complex dendritic structures, they adhere more tightly to the culture dish and the digestion time is also extended (3 minutes). The specific steps for cell preparation are as follows: Figure 4 shown.

[0138] (3) Establishment of RT4 animal model

[0139] Digest and resuspend RT4 cells in the logarithmic growth phase in 5 mL PBS, take 10 μL to count the cells, and adjust the viable cell concentration to 1 × 10 4 After the SD rats were anesthetized, they were mask-anesthetized and fixed on a constant temperature stage. The skin was prepared and scribed according to the aseptic principle. The skin and muscles were cut layer by layer. The sciatic nerve was exposed through the intermuscular space. The sciatic nerve was freed and stabilized with microtweezers or a dissector. The RT4 cell suspension was slowly injected under a microscope. The injection was maintained for 1 minute and the needle was slowly withdrawn. The specific operation process and schematic diagram are shown in the figure. Figure 5 shown.

[0140] For the rat trigeminal nerve RT4 tumor model, the concentration of live cells needs to be adjusted to 5×10 4 / uL, the positioning point is the rat's bregma as the origin (X=0, Y=0, Z=0), the skull drilling position is (X=-3.78mm, Y=-3.23mm), the depth of the microinjector needle is Z=11.50mm, and the needle reaches the skull base before reaching 11.50mm. If the needle cannot be inserted further, the point where it reaches the skull base is the injection point. The injection speed is 0.5uL / min, the injection volume is 2uL, and the needle is stopped for 4 minutes after injection and then slowly withdrawn.

[0141] (III) Establishment of the U87MG tumor cell animal model in BALB / c nude mice

[0142] (1) Culture of U87MG cell line

[0143] The U87MG cell line was maintained at the Chengdu Tianfu Life Science Park. After thawing, the cells were allowed to adhere to the culture dish. The remaining procedures were similar to those for the C6 cell line. Once the cells stabilized, they were used to model brain gliomas in nude mice.

[0144] (2) U87MG cell preparation

[0145] The concentration of U87MG live cells was adjusted to 10 4 The remaining operations are the same as those for C6 cell preparation. The specific operation procedures are as follows: Figure 6 shown.

[0146] (3) Establishment of U87MG animal model

[0147] The rat fixator on the Rayward stereotaxic instrument was replaced with a mouse fixation adapter to fix the nude mice and maintain anesthesia. The injection point was (X = -1.00 mm, Y = -1.34 mm, Z = 3.00 mm), and the injection volume of the cell suspension was 10 μL. The remaining operations were the same as those for rat stereotaxic intracranial tumor implantation. The specific operation process is as follows: Figure 7 shown.

[0148] (IV) Establishment of IOMM-Lee tumor cell animal model in BALB / c nude mice

[0149] (1) Culture of IOMM-Lee cell line

[0150] The IOMM-Lee cell line was maintained at the Chengdu Tianfu Life Science Park. After thawing, the cells were allowed to adhere to the culture dish. The remaining procedures were similar to those for the C6 cell line. Once the cells stabilized, they were used to model sciatic meningioma in nude mice.

[0151] (2) Preparation of IOMM-Lee cells

[0152] Adjust the concentration of IOMM-Lee live cells to 10 4 The rest of the operation is the same as that of C6 cell preparation. The specific operation process is as follows: Figure 8 shown.

[0153] (3) Establishment of the IOMM-Lee animal model

[0154] The rat fixator on the Rayward stereotaxic instrument was changed to a mouse adapter to fix the nude mice and maintain anesthesia. The injection volume of the cell suspension was 10uL. The remaining operations were the same as those for SD rat sciatic nerve RT4 tumor implantation. The specific operation process is as follows: Figure 9 shown.

[0155] 1.2 Application of OCT system in tumor boundary detection in living animal models

[0156] All tumor-bearing animals undergo OCT system detection of the tumor boundary of the nervous system in the live animal tumor-bearing model 2-3 weeks after tumor implantation to verify the effectiveness and scientificity of the system. First, the animal is anesthetized by induction with isoflurane. After satisfactory anesthesia, it is immediately fixed in a stereotaxic apparatus so that the mouse's teeth bite into the central fixation rod and the mouse's external auditory canals are fixed. Anesthesia is maintained with isoflurane. For intracranial tumor-bearing animal models, the skin is prepared, routine disinfection is performed, and the scalp is incised according to aseptic principles. The skin is retracted with a retractor, and a bone window of appropriate size is formed using a drill, bone saw, etc. to expose the lesion area. If necessary, the bone window can be enlarged to fully expose the bilateral frontal, temporal, parietal, occipital, and cerebellum. Hemostasis is carefully stopped using bipolar electrocoagulation, micro-gauze, cotton ball compression, and other hemostatic methods. The dura mater is opened. For supratentorial gliomas, an OCT probe is used to probe the brain tissue surface before incising the brain tissue. Considering that the effective probing depth of the OCT system for brain tissue is 2-3 mm, after the OCT system has scanned the brain tissue surface, the OCT scan number is recorded. Simultaneously, a Leica microscope is used to photograph the probing area to record and capture the surgeon's naked-eye observations. A transverse section of tissue approximately 2 mm thick and of appropriate length and width is cut along the axial plane using an ultra-optimal knife. The probing surface is marked with a fuchsin solution (to facilitate determination of the cutting direction for subsequent pathological sections). Immediately, the specimen is fixed with 4% paraformaldehyde in a cryovial or centrifuge tube and labeled with the number, specimen location, and characteristics. Subsequently, tissue morphology is observed at 2-mm intervals, and the region of interest (ROI) is subjected to in vivo OCT scanning. Simultaneously, tissue from the probing area is removed, and the probing surface is demarcated with a fuchsin solution. In animal models of central nervous system (CNS) brain tumor invasion, including the C6 and U87 brain tumor models, an OCT system is used to scan the tumor layer by layer during surgery. If only tumor tissue is visible in grayscale OCT images within the scanned layer, without peritumoral normal brain tissue, the tumor is resected under a microscope, labeled with magenta, and fixed with 4% paraformaldehyde before being removed. The OCT system is then used again, repeating the above steps. This allows for real-time intraoperative OCT monitoring to determine if the resection will reach the tumor boundary. When the residual thickness of the resected tumor is less than 3 mm, the OCT system can detect normal brain tissue or cranial nerves deep within the tumor, providing a warning of potential problems. At this point, patient debulking is continued under the microscope, resecting approximately 2 mm of tumor tissue and continuously reducing the residual tumor thickness to less than 1 mm. After further resection of the residual tumor, the OCT system is used to monitor the tumor cavity in real time during surgery. If the OCT system imaging shows that the residual tumor thickness in a certain detected area is greater than 1mm, careful resection is performed to reduce the residual tumor thickness to a minimum (less than 1mm). In short, under the guidance of the real-time intraoperative detection of the living tumor by the OCT system, tumor reduction operations are performed to control the residual tumor thickness in most areas of the tumor cavity to less than 1mm.When the OCT system showed that the residual tumor in most areas of the tumor cavity was controlled within 1 mm, the tumor cavity specimen in the detection area was obtained and fixed.

[0157] For the trigeminal nerve and sciatic nerve infiltrated adhesion tumor-bearing animal models, a similar operating procedure is used. After the tumor is exposed, OCT detection is performed before tumor resection. The OCT probe is used to detect and scan each layer approximately every 2 mm. The Leica microscope records the surgeon's observations. A super-optimal knife is used to obtain neat specimens with a 2 mm layer thickness. The OCT detection surface is marked with magenta and fixed with 4% paraformaldehyde. During the tumor resection process, the sciatic nerve or trigeminal nerve located deep in the tumor (<3 mm) is identified under the real-time detection and indication of the OCT system to provide an early warning for the area near the tumor boundary, reminding the surgeon to operate gently to minimize the traction of the peripheral nerves and ultimately completely remove the tumor.

[0158] In animal models with tumors on the sciatic nerve, the tumors are mostly spherical, and most of the tumors are located below the nerve, with only a 1-2mm layer of tumor wrapped above the nerve. To facilitate the OCT system to detect the ventral side of the tumor and nerve, and to simulate the scenario of predicting the course of the facial nerve during microsurgical resection of acoustic neuroma, the distal end of the sciatic nerve is transected to facilitate multi-angle detection of the tumor and nerve, layer-by-layer scanning, resection and specimen sampling. At the same time, the unilateral severance method ensures that the blood supply to the tumor is not completely interrupted, so as to better simulate the process of acoustic neuroma resection in the human body.

[0159] 1.3 Pathological Verification of the OCT System Detection Area

[0160] During the OCT system detection and tumor resection process, paraffin sections of supratentorial glioma and peritumoral normal brain tissue, glioma and peritumoral normal cerebellum tissue, glioma and peritumoral normal brainstem tissue, glioma and peritumoral normal basal ganglia tissue, sciatic nerve sheath tumor, and trigeminal nerve sheath tumor of SD rats were prepared; paraffin sections of human brain glioma and peritumoral normal brain tissue, and sciatic meningioma of BALB / c nude mice were prepared and HE staining was performed. The specific operation steps are as follows Figure 10-11 .

[0161] 2. Application of OCT system in intraoperative boundary detection in tumor-bearing animal models

[0162] 2.1 Tumor formation effect and OCT detection data

[0163] Among the 5 SD rat supratentorial glioma models, 1 died on the day of tumor cell implantation, and the remaining 4 developed tumors. Among the 8 SD rat brainstem glioma models, 7 rats developed tumors, and 1 rat died on the day of implantation. Among the 2 SD rat cerebellar glioma models, 2 rats developed tumors. Among the 7 SD rat chiasmatic glioma models, 1 rat died during implantation due to an anesthesia accident, and the remaining 6 rats developed tumors. Among the 11 sciatic nerve sheath tumor models, 8 rats developed tumors, and 3 did not develop tumors macroscopically. Among the 4 trigeminal nerve sheath tumor models, 2 rats developed tumors, and 2 did not develop tumors macroscopically. Among the 7 nude mouse supratentorial glioma models, 1 rat died during implantation due to an anesthesia accident, and the remaining 6 rats developed tumors. Among the 6 nude mouse sciatic nerve meningioma models, 6 rats developed tumors.

[0164] The OCT system was used to examine a total of 45 in vivo tumor-bearing animal models, including 4 supratentorial C6 rats, 7 brainstem C6 rats, 2 cerebellar C6 rats, 11 sciatic RT4 rats, 3 trigeminal RT4 rats, 6 supratentorial U87 nude mice, and 6 sciatic IOMM nude mice. A total of 182 in vivo specimens were examined, including 59 specimens at the junction of tumor and normal brain tissue, 63 specimens at the junction of tumor and peripheral nerve, and 16 specimens at the tumor cavity to detect residual tumor.

[0165] 2.2 Application of OCT system in intraoperative boundary detection of intracranial glioma in SD rats

[0166] 4 cases of supratentorial brain parenchymal gliomas were detected by the OCT system; 6 cases of optic chiasmatic gliomas were detected by the OCT system; 7 cases of brainstem gliomas were detected by the OCT system; 2 cases of cerebellar gliomas were detected by the OCT system. Figures 12 to 29 As shown. After detection, the tissue type of the detection area was pathologically verified by HE staining. According to the imaging performance of the OCT scanning grayscale image, the grayscale image of C6 glioma showed signs of low density or slightly low density shadows, and the normal brain tissue peritumoral (including supratentorial normal brain tissue, brainstem and cerebellum tissue) showed signs of high density or slightly high density shadows: the two-dimensional color image reconstructed by ImageJ showed that C6 glioma had imaging characteristics with mainly low color levels (green, yellow, red) compared with the normal brain tissue peritumoral area, and the normal brain tissue peritumoral had imaging characteristics with mainly higher color levels (blue, green or yellow). The optic nerve also showed an imaging manifestation of high density shadows in the OCT scanning grayscale image. According to the OCT imaging differences between the tumor and the normal tissue peritumoral (brain parenchyma or optic nerve), the OCT system can be used to assist in real-time identification of the types of tumor and normal brain tissue peritumoral during surgery (such as Figure 13-14 、 Figure 19 、 Figures 26-27): When there is a thin layer of residual tumor (about 2mm), the normal brain tissue or optic nerve located deep in the thin layer of tumor is detected to play an early warning role for the deep normal tissue and optic nerve (such as Figure 15-17 、 Figures 19-23 、 Figure 28-Figure 29 ): After the tumor is resected, the OCT system is used again to detect the tumor cavity to verify whether there is residual tumor (such as Figure 24-25 shown).

[0167] like Figures 12 to 14 As shown, the SD rat supratentorial C6 glioma animal model provided by the embodiment of the present invention, the preoperative head magnetic resonance T2 image ( Figure 12 ) shows a mass in the right frontal lobe (two-angled arrow). After the animal is induced with anesthesia, it is properly fixed and anesthesia is maintained with a mask. Corresponding ECG monitoring clips and blood oxygen saturation detection clips are attached to the limbs, and a rectal temperature monitor is inserted into the anus. The ECG monitor displays the animal's basic vital signs such as the ECG waveform, heart rate, blood oxygen saturation, respiratory rate and body temperature. During the operation, anesthesia parameters such as isoflurane concentration and oxygen flow are adjusted according to the numerical changes to more safely perform in-vivo tumor boundary detection. After craniotomy, the bone window is removed, the brain tissue covering the tumor surface is removed, the tumor area is exposed, and the area of ​​interest (green box) is detected in real time by the OCT system without contact. A B-scan grayscale image of the area ( Figure 13 ) shows the boundary between the tumor and the normal brain tissue around the tumor (orange dotted line). The left side of the dotted line is the low-density area, corresponding to the tumor area, and the right side of the dotted line is the high-density area, corresponding to the normal brain tissue area around the tumor. The two-dimensional color image reconstructed using ImageJ software ( Figure 14 ) shows that the tumor area is mainly low-level (green), and the normal brain tissue around the tumor contains higher-level (blue).

[0168] like Figures 15 to 17 As shown in the optic chiasm C6 tumor-bearing SD rat animal model provided by the embodiment of the present invention, the preoperative head MRI T2 image showed that the glioma originated from the optic chiasm and compressed the bilateral basal ganglia and ventricular system. After resection of most of the bilateral frontal lobes, the bilateral optic nerves and the gliomas originating from the optic nerves were visible. The OCT system scanned the region of interest, and the surface grayscale image ( Figure 15 ) shows the bilateral optic nerves above, indicating that the region of interest is accurately located. A B-scan grayscale image ( Figure 16 ) shows the high- and low-density junction area (orange dotted line). The low-density area on the upper right is the glioma, and the high-density area on the lower left is the optic nerve, thus showing the boundary between the glioma and the optic nerve. Reconstructed two-dimensional color image ( Figure 17 ) shows that the tumor area (black box) is mainly low-level (yellow and red), and the exposed optic nerve and the optic nerve covered by glioma (white oval circle) both show imaging characteristics mainly in high-level (green), and the contrast is more obvious in the boundary area between the two.

[0169] like Figures 18 to 25 As shown, the brainstem C6 tumor-bearing SD rat animal model provided by the embodiment of the present invention, preoperative MRI plain scan T2 image ( Figure 18 ) showed that the tumor was located in the right medulla oblongata and dorsal medulla oblongata. After cutting the bilateral cerebellum, the tumor and brainstem area were exposed. The grayscale image generated by OCT detection ( Figure 19 ) shows a blocky low-density area (*) covering the slightly higher-density area below (arrow), indicating that the tumor covers the brainstem and shows the boundary between the glioma and the brainstem. Part of the tumor was removed to thin the tumor tissue on the surface of the brainstem, and OCT detection was performed again. The grayscale image ( Figure 20 ) shows that the tumor area (*) is a low-density area, and the peritumoral area is a high-density area (arrow), indicating that it is the brainstem tissue around the tumor. Reconstructed two-dimensional color visualization ( Figure 21 ) shows that there is a high-level (blue) attachment (arrow) phenomenon on the surface of the brainstem area relative to the tumor area (*). Continuing to remove the tumor covering the surface of the brainstem, a small piece of tumor tissue was found in the medulla oblongata. The OCT grayscale image ( Figure 22 ) and two-dimensional color map ( Figure 23 ) shows that the tumor area (arrow) presents low density and low color scale images, while the normal brainstem tissue around the tumor (#) presents high density and high color scale images. The glioma mass located in the medulla oblongata was carefully removed, and the tumor cavity was detected by OCT. The grayscale image ( Figure 24 ) and two-dimensional color map ( Figure 25 ) showed that the tumor cavity was high-density signal and high color level, indicating that the detected area was likely to be normal brainstem tissue around the tumor, and the possibility of residual glioma in the tumor cavity was low.

[0170] like Figures 26 to 29 As shown in the figure, the cerebellar C6 tumor-bearing SD rat animal model provided by the embodiment of the present invention was partially removed and then detected using an OCT system to verify whether the system can assist the surgeon in distinguishing the boundary between the glioma and the normal cerebellum around the tumor. A B-scan grayscale image ( Figure 26 ) shows the low-density area of ​​glioma (red dashed box) and the boundary between glioma and cerebellum tissue (arrow). Reconstructed two-dimensional color image ( Figure 27 ) shows that the surface of the normal tissue area contains a thin layer of light blue area (arrow). After further resection of the glioma, the OCT system was used to detect the tumor boundary, and the B-scan grayscale image ( Figure 28 ) A thin layer of high-density shadow (arrow) is seen, and the low-density shadow area (two red irregular dotted areas) surrounding it indicates the location of the tumor. Two-dimensional color image ( Figure 29) shows that the surface peritumoral normal cerebellum tissue has high-level (blue) enriched signs (black arrows), while the slightly deeper peritumoral normal cerebellum tissue has higher-level (green) enriched signs (white arrows) than the upper adjacent tumor area (black irregular dotted area) (mainly yellow and red).

[0171] 2.3 Application of OCT system in intraoperative boundary detection of schwannoma in SD rats

[0172] 8 cases of sciatic nerve RT4 tumors were detected, 3 cases were not detected by naked eyes, and 11 cases were detected by OCT system. 2 cases of trigeminal nerve RT4 tumors were detected, 2 cases were not detected by naked eyes, and 3 cases were detected by OCT system. Figures 30 to 38 In the RT4 sciatic nerve sheath tumor model, since the tumor grows in a short period of time, most of the sciatic nerve capsule is still intact. Therefore, the position and adhesion relationship between the tumor and the sciatic nerve in this tumor model is similar to the relationship between the tumor and the facial nerve in patients with acoustic neuroma. The OCT system is used to detect the living tissue specimens of the schwannoma animal model. After detection, the pathological properties of the detected area are verified by HE staining. Schwannoma appears as a low-density shadow in the OCT grayscale image ( Figure 30 ), the surface of the sciatic nerve contains a perifascicle, which is shown in the OCT grayscale image as a thin layer of high-density shadow on its surface ( Figure 32 , Figure 34 , Figure 35 , Figure 36 , Figure 37 This can be used to identify nerve tissue deep within the schwannoma (2-3 mm), providing an early warning of nerves deep within the tumor.

[0173] The tumor formation effect of RT4 on the right sciatic nerve of SD rat No. 17 provided in the embodiment of the present invention shows a spherical tumor. The sciatic nerve runs deep in the tumor. One of the B-scan images of the region of interest is shown in FIG. Figure 30 As shown, the tumor area is a uniform low-density shadow, and the ImageJ software reconstructs the two-dimensional color image ( Figure 31 ) shows that the tumor tissue in the detection area is uniformly green or yellow. The tumor is removed, leaving a thin layer of tumor tissue in the nerve (1-2mm). Due to the deep detection depth, the distal sciatic nerve is cut and placed in a superficial position. A B-scan grayscale image (orange line segment) of the region of interest (green frame) shows a slightly higher density layer below the low density shadow ( Figure 32 , arrow), consistent with the OCT imaging manifestation of the sciatic nerve perineurium. The slightly high-density shadow is below the sciatic nerve covered by the schwannoma, which shows the boundary between the schwannoma and the sciatic nerve: two-dimensional color image ( Figure 33) shows a green layered signal shadow (arrow) with the same color scale as the surface layer. The figure shows the effect of RT4 on the right sciatic nerve of SD rat No. 42. The figure shows a spindle-shaped thin layer tumor. An OCT scan was performed along the cross section of the nerve (green box). A B-scan grayscale image ( Figure 34 ) shows that the perineurium of the sciatic nerve (arrow) appears as a thin layer of high-density shadow: OCT scan of the distal sciatic nerve tumor and nerve complex, a B-scan grayscale image ( Figure 35 ) shows a low-density shadow (corresponding to the schwannoma), and a thin layer of high-density shadow in the deep surface (corresponding to the sciatic nerve membrane), indicating the boundary between the schwannoma and the sciatic nerve. The image of the tumor formed by RT4 on the right sciatic nerve of SD rat No. 45 shows that the tumor envelops the sciatic nerve. The OCT system scans the region of interest (green box) longitudinally along the direction of the nerve. The B-scan grayscale image ( Figure 37 ) shows a continuous thin layer of high-density shadow (arrow), and the two-dimensional color image ( Figure 38 ) shows a thin layer of high signal (blue color scale) shadow (arrow), indicating that the area is the perineurium of the sciatic nerve exposed and covered by the schwannoma. The tumor was removed, leaving only a thin layer of tumor remaining in the sciatic nerve. The B-scan grayscale image of the OCT scan ( Figure 36 ) shows the nerve and tumor boundary (arrow).

[0174] 2.4 Application of OCT system in intraoperative boundary detection of glioma in BALB / c nude mice

[0175] Six BALB / c nude mice were diagnosed with supratentorial gliomas, and all six were examined with the OCT system. The OCT system was used to examine the living tissue specimens of the nude mouse glioma model, and the tissue type of the examined area was verified by HE staining. Figures 39 to 44 Similar to SD rats, the OCT scanning system can help surgeons distinguish between human glioma and normal brain tissue around the tumor ( Figure 39-40 ), search for residual tumor in the tumor cavity ( Figure 41-42 ) and confirm whether the tumor is completely removed ( Figure 43-44 ).

[0176] The present invention provides an example of a right supratentorial U87 nude mouse. After the tumor is exposed, the OCT system is used to detect the tumor and the normal brain tissue boundary around the tumor. A B-scan grayscale image ( Figure 39 ) shows the boundary (orange dashed line) between the tumor (low-density shadow area) and the normal brain tissue (high-density shadow area): 2D color reconstruction image ( Figure 40 ) shows that the normal brain tissue area around the tumor (#) has thicker high-color (blue, green) imaging appearance than the tumor area (*). The tumor was partially removed, leaving part of the tumor at the bottom of the tumor cavity, and OCT detection was performed. A B-scan grayscale image ( Figure 41) showed that the bottom of the tumor cavity was low signal and the two sides of the tumor cavity were slightly high signal, which was consistent with the imaging manifestation that the bottom of the tumor cavity was a tumor and the two sides of the tumor cavity were normal brain tissue. Figure 42 The bottom surface of the tumor cavity (*) has less high-level (blue) enrichment than the sides of the tumor cavity. After further resection of the residual tumor at the bottom of the tumor cavity, OCT detection was performed, and the B-scan grayscale image ( Figure 43 ) and the corresponding two-dimensional color map ( Figure 44 ) showed that both sides and the bottom of the tumor cavity were high-density and high-signal shadows, which were consistent with the OCT imaging manifestations of normal brain tissue, indicating that the tumor had been completely removed.

[0177] Application of 2.5OCT system in intraoperative border detection of meningioma in BALB / c nude mice

[0178] Six mice developed sciatic meningiomas, and all six were examined with the OCT system. The OCT system was used to examine the living tissue of the nude mouse sciatic meningioma animal model during surgery, and the tissue type of the examined area was verified by HE staining after examination. Figure 45-46 shown.

[0179] In the present invention, an example of a nude mouse with a right sciatic nerve IOMM tumor was provided. The tumor covering the surface of the sciatic nerve was removed until only a thin layer of tumor remained. The OCT system was used for detection. The grayscale image ( Figure 45 ) shows that the sciatic nerve is a tubular high-density shadow (arrow), with a strip-shaped low-density shadow below it and the surrounding tumor tissue is a low-density shadow. Figure 46 ) shows that the surface of the sciatic nerve is enriched with high-level signals, while the surrounding area is mainly low-level, thus showing the boundary between meningioma and sciatic nerve.

[0180] 2.6 Data Analysis of OCT Boundary Detection in Live Tumor-Bearing Animal Models

[0181] (1) Grid method

[0182] The OCT system was used to detect in vivo a total of 71 tumors and 71 normal peritumoral tissues that had been pathologically verified. The sum of the pixels with grayscale values ​​between 100 and 140 within a 55*80 (width*height) grid was measured using the grid method. The relevant statistical results are shown in Table 1.

[0183] Table 1 The sum of the grid pixel points of OCT in vivo detection of tumors and normal tissues around tumors in animal experiments *Statistical results

[0184]

[0185] * Refers to the sum of pixels with grayscale values ​​between 100 and 140 within a 55*80 (width*height) grid.

[0186] In 15 cases of supratentorial C6 tumors and peritumoral normal brain tissue, the average value of the tumor area was 92.47±198.25, and the average value of the peritumoral normal brain tissue was 472.33±372.51. The independent sample t-test showed that there was a statistically significant difference between the two (P=0.002).

[0187] In 12 cases of optic chiasm C6 tumors and optic nerves, the average value of the tumor area was 87.58±101.27, and the average value of the normal brain tissue around the tumor was 401.25±309.54. The independent sample t-test showed that there was a statistically significant difference between the two (P=0.005).

[0188] In 16 cases of brainstem C6 tumors and peritumoral normal brainstem tissues, the average value of the tumor area was 101.37±93.55, and the average value of the peritumoral normal brain tissue was 393.69±179.73. The independent sample t-test showed that there was a statistically significant difference between the two (P<0.001).

[0189] In 6 cases of C6 tumors and peritumoral basal ganglia tissues, the average value of the tumor area was 141.50±60.07, and the average value of the peritumoral normal cerebellum tissue was 482.33±162.60. The independent sample t-test showed that there was a statistically significant difference between the two (P=0.001).

[0190] In 8 cases of cerebellar C6 tumors and peritumoral normal cerebellar tissue, the average value of the tumor area was 144.75±214.30, and the average value of the peritumoral normal cerebellar tissue was 435.38±347.76. The independent sample t-test showed no statistical difference between the two (P=0.064).

[0191] In 14 cases of supratentorial U87 tumors and peritumoral normal brain tissue, the average value of the tumor area was 64.36±60.44, and the average value of the peritumoral normal brain tissue was 512.29±273.04. The independent sample t-test showed that there was a statistically significant difference between the two (P<0.001).

[0192] (2) Average gray value method

[0193] A total of 71 tumors and 72 normal peritumoral tissues detected by the OCT system and verified by pathology were extracted. The average grayscale value algorithm was used to calculate the average grayscale value within a specific width and depth range (41 rows). The statistical results are shown in Table 2.

[0194] Table 2 Statistical results of the average grayscale values ​​of OCT in vivo detection of tumors and normal tissues around tumors in animal experiments

[0195]

[0196] In 13 cases of supratentorial C6 tumors and peritumoral normal brain tissue, the mean grayscale value of the tumor region was 58.73±16.67, and that of the peritumoral normal brain tissue was 74.34±12.95. An independent sample t-test showed a statistically significant difference between the two (P=0.014).

[0197] In 12 cases of optic chiasm C6 tumors and optic nerves, the average grayscale value of the tumor area was 45.30±14.09, while that of the normal peritumoral brain tissue was 59.23±14.60. An independent sample t-test showed a statistically significant difference between the two (P=0.027).

[0198] In 17 cases of brainstem C6 tumors and peritumoral normal brainstem tissue, the average grayscale value of the tumor area was 59.46±11.34, and that of the peritumoral normal brain tissue was 71.11±13.24. An independent sample t-test showed a statistically significant difference between the two (P=0.010).

[0199] In seven cases of basal ganglia C6 tumors and peritumoral normal basal ganglia tissue, the average grayscale value of the tumor region was 64.37±13.26, and that of the peritumoral normal brain tissue was 79.16±14.83. An independent sample t-test showed no statistically significant difference between the two (P=0.073).

[0200] In 7 cases of cerebellar C6 tumors and peritumoral normal cerebellar tissue, the mean grayscale value of the tumor region was 57.94±8.56, and that of the peritumoral normal brain tissue was 64.24±22.69. An independent sample t-test showed no statistical difference between the two (P=0.512).

[0201] In 15 cases of supratentorial U87 tumors and 16 cases of peritumoral normal brain tissue, the mean grayscale value of the tumor region was 61.31±16.47, and that of the peritumoral normal brain tissue was 76.71±13.17. An independent sample t-test showed a statistically significant difference between the two (P=0.007).

[0202] (3) Statistical description method

[0203] In eight SD rats with RT4 sciatic nerve tumors, the OCT system was able to detect the sciatic nerve and its perineurium deep within the schwannoma (<3 mm), appearing as a thin tubular or crescent-shaped shadow with a higher density than the surrounding area. Reconstructed grayscale and two-dimensional color images showed high-level enrichment of the nerve and its perineurium covered by the tumor, highlighting the boundary between the schwannoma and the sciatic nerve.

[0204] In the 10MM animal model of the sciatic nerve of six nude mice with tumors, the sciatic nerve of one nude mouse was severely damaged by tumor erosion, and the sciatic nerve was not observed during the entire process of tumor resection. The OCT system was able to detect the sciatic nerve and its perineurium located deep within the meningioma (approximately 1-2 mm) in the remaining five nude mice. This appeared as a thin tubular shadow or crescent shadow with a higher density than the surrounding area, and a crescent shadow with a higher density than the surrounding area was visible in the OCT grayscale image. In addition, the grayscale two-dimensional color image showed that the nerve and its perineurium covered by the tumor showed a high-level enrichment phenomenon, which could show the boundary between the meningioma and the sciatic nerve.

[0205] For the two SD rat trigeminal nerve RT4 tumor-forming animal models, the OCT system was able to detect the schwannoma tissue and the trigeminal nerve around the tumor. The schwannoma tissue showed a lower density imaging feature on the grayscale image than the trigeminal nerve, showing the boundary between the schwannoma and the trigeminal nerve.

[0206] The significance of 3OCT system in tumor boundary detection in living tumor-bearing animal models

[0207] Detecting tumor boundaries using OCT in living animals is crucial. Loss of blood perfusion and volumetric deformation during specimen acquisition can lead to discrepancies between the OCT grayscale values ​​of ex vivo specimens and those of living specimens. Furthermore, surgical interference with the detection area, such as intraoperative bleeding and hematoma formation, scab formation after bipolar coagulation, and the regular pulsation of brain tissue, can affect the OCT system's detection process and the resulting grayscale image.

[0208] A review of literature reporting on the use of OCT systems for neurological tumors reveals that the tumor-bearing animal models involved include supratentorial glioma models and cerebellar glioma models. However, there are no reports of using OCT systems to detect tumor boundaries in the brainstem glioma, optic nerve glioma, sciatic nerve sheath tumor, trigeminal nerve sheath tumor, or peripheral nerve meningioma models established in this paper.

[0209] The present invention establishes two major categories of tumor-bearing animal models. One type is an infiltrative growth animal model, including a rat brain glioma model, a rat optic chiasmatic glioma model, and a nude mouse human brain glioma model. The other type is an adhesive growth animal model, including a rat peripheral nerve malignant schwannoma model (including a trigeminal schwannoma model and a sciatic schwannoma model) and a nude mouse peripheral nerve malignant meningioma model. These models simulate clinical disease models of human brain parenchymal gliomas, optic pathway gliomas, schwannomas, and meningiomas, verifying the effectiveness and practicality of the OCT system in detecting the boundaries between gliomas and brain tissue, gliomas and optic nerves, schwannomas and peripheral nerves, and meningiomas and peripheral nerves.

[0210] For infiltrative tumor models, the OCT system can detect and differentiate glioma tissue and peritumoral normal brain tissue and optic nerve. It can also play an early warning role for peritumoral normal brain tissue and optic nerve located deep in the tumor (within 2-3 mm). It can also detect the residual tumor in the tumor cavity after tumor resection to minimize the amount of residual tumor. Among them, the application of the OCT system in the detection of brainstem glioma boundaries and the application of the OCT system in the detection of the boundaries of glioma and peritumoral normal basal ganglia when supratentorial glioma invades the basal ganglia are particularly meaningful. The main reasons include the following three points: (1) The feasibility and difficulty of conducting relevant research in humans. Even if the OCT system is used to detect brainstem glioma and the relevant areas of glioma invading the basal ganglia during surgery, and OCT grayscale image data is obtained, it is impossible to obtain specimen tissue of important functional areas and obtain pathological confirmation that the detected area is the brainstem or basal ganglia. Therefore, it is particularly important to obtain OCT grayscale image data of the tumor and peritumoral normal brainstem and basal ganglia tissue in animal experiments, and obtain pathological confirmation at the same time. (2) The detection of glioma boundaries in this area has extremely important clinical significance. The surgical treatment of intracranial tumors should reflect precise surgical treatment. For brainstem gliomas and gliomas invading the basal ganglia, precise surgical treatment is even more important because it involves the protection of the neurological function of important functional areas. The present invention proves that the OCT system can assist in the intraoperative identification of tumor boundaries in animal models of brainstem gliomas and gliomas invading the basal ganglia, thereby proving that the OCT system has the ability to assist in precise surgical treatment of gliomas in important neurological functional areas. (3) Lack of research in domestic and foreign literature. There are no reports on the application of OCT systems to the detection of boundaries in animal models of brainstem gliomas and gliomas invading the basal ganglia in the relevant literature at home and abroad. This may be because the detection site is deep, and it is difficult to perform craniotomy to remove the tumor in this site. At the same time, it is difficult to carry out in vivo detection with the OCT system. Therefore, it further reflects the significance and value of the present invention in systematically carrying out the application of OCT systems to the detection of boundaries of various tumors.

[0211] For adhesive growth tumor models, the OCT system can detect and differentiate between gliomas and the optic nerve, neurilemmomas and the sciatic and trigeminal nerves, and meningiomas and the sciatic nerve: it can provide early warning for the optic nerve, sciatic nerve, and trigeminal nerve located deep within the tumor (within 2-3 mm), prompting the surgeon to operate gently, reduce the traction on the nerves, and maximize the likelihood of preserving nerve function. The present invention believes that the role of the OCT system in adhesive growth tumor models is of special significance. A review of the literature shows that the application of the OCT system in intracranial adhesive growth tumor models has not yet attracted the attention, research, and reports of researchers. The conclusions drawn by the present invention, especially the early warning role of the OCT system for deep peripheral nerves in adhesive tumor models, demonstrate that the OCT system can not only detect tumor boundaries in invasive tumor models, but also has the potential to identify tumor boundaries in adhesive tumor models during surgery. Unlike neuroelectrophysiological monitoring technology used to determine the course of cranial nerves during surgery, its accuracy depends on direct stimulation of the nerves after exposure. For cranial nerves deeply buried on the ventral side of the tumor tissue or wrapped in the tumor, the sensitivity and specificity of electrophysiological detection are greatly reduced. However, the present invention proves that the OCT system can indicate and warn the course of cranial nerves when the cranial nerves are still located deep in the tumor. This suggests that the OCT system is expected to be similar to neuroelectrophysiological monitoring technology, but with the unique advantage of deep-surface warning function, and has the potential to become an alternative technology for real-time monitoring of the course of cranial nerves in the tumor area during surgery. It also suggests that continuing to improve the performance of the OCT system, especially the improvement in detection depth, will play a positive role in enhancing the boundary warning capability of adhesion tumors and improving the application value.

[0212] In vivo detection of tumor-bearing animal models, OCT systems can perform real-time intraoperative detection. Grayscale images of tumor tissue (gliomas, schwannomas, and meningiomas) often appear as low-density areas, while normal tissue (brain tissue and peripheral nerves) often appear as high-density areas. Therefore, OCT systems can assist surgeons in distinguishing gliomas from normal peritumoral brain tissue (including supratentorial brain tissue, cerebellum, and brainstem), gliomas from the optic nerve, schwannomas from peripheral nerves (including the sciatic and trigeminal nerves), and meningiomas from the sciatic nerve. OCT systems have the potential to become a complementary technology for detecting the boundaries of nervous system tumors.

[0213] In order to prove the creativity and technical value of the technical solution of the present invention, this section provides application examples of the claimed technical solution on specific products or related technologies.

[0214] Pages 18 to 27 of the present invention are all examples of actual living tumor-bearing animal models used to verify the OCT system, which can reflect the creativity and technical value of the technical solution of the present invention.

[0215] The embodiments of the present invention have achieved some positive results during the development or use process, and indeed have great advantages over the existing technology. The following content describes them in conjunction with data, charts, etc. from the experimental process.

[0216] Special note: All the pictures and two tables in this application reflect the positive effects of the embodiments of the present invention during research and development or use, and indeed have great advantages over the existing technology. For example, the existing technology of using human ex vivo brain tissue or tumor tissue specimens for OCT system measurement cannot obtain normal tissue of important functional areas, such as brainstem and basal ganglia, while tumor-bearing animal models can achieve the acquisition of normal tissue in these areas, which has great advantages.

[0217] The above description is only a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications, equivalent substitutions and improvements made by any technician familiar with this technical field within the technical scope disclosed by the present invention and within the spirit and principles of the present invention should be covered by the scope of protection of the present invention.

Claims

1. A method for establishing a tumor-bearing animal model for an OCT system, characterized in that: include: The C6 cell line was used to establish tumor-functional brain parenchyma and brain nerve infiltration models in SD rats, including the SD rat brainstem glioma model, basal ganglia glioma model, and optic chiasm glioma model; SD rat tumor-nerve adhesion models were established using the RT4 cell line, including the sciatic nerve RT4 tumor model and the trigeminal nerve RT4 tumor model; The U87MG cell line was used to establish BALB / c nude mouse models of tumor-brain parenchymal invasion, including a BALB / c nude mouse brainstem glioma model and a basal ganglia glioma model; and the IOMM-Lee cell line was used to establish a BALB / c nude mouse model of tumor-sciatic nerve adhesion, namely the IOMM-Lee tumor cell animal model. The method for establishing a tumor-bearing animal model for an OCT system comprises the following steps: Step 1: C6 animal models were constructed by culturing C6 cell lines and preparing C6 cells to obtain supratentorial parenchymal glioma models, chiasmatic glioma models, and brainstem glioma models in SD rats. Step 2: culturing the RT4 cell line, preparing RT4 cells, and establishing an RT4 animal model, thereby establishing a rat sciatic nerve sheath tumor model and a rat trigeminal nerve RT4 tumor model to obtain an SD rat RT4 tumor cell animal model; Step 3: Cultivate the U87MG cell line, prepare U87MG cells, and establish a U87MG tumor cell animal model in BALB / c nude mice; Step 4: Cultivate the IOMM-Lee cell line, prepare IOMM-Lee cells, and establish a BALB / c nude mouse IOMM-Lee tumor cell animal model; The culturing of the IOMM-Lee cell line, the preparation of IOMM-Lee cells, and the establishment of a BALB / c nude mouse IOMM-Lee tumor cell animal model include: 1) IOMM-Lee cell line culture: IOMM-Lee cells were cultured in a 37°C, 5% CO2 incubator for 24 hours. The medium was changed based on cell growth and culture medium color change. When cells covered 80%-90% of the flask bottom area, the IOMM-Lee cells were passaged at a ratio of 1:2 to 1:

3. After passage, the IOMM-Lee cells were cultured in high-glucose DMEM medium supplemented with 10% fetal bovine serum in a 37°C, 5% CO2 incubator. The culture medium was changed every 1-2 days based on cell growth, and cell growth was observed and recorded daily. IOMM-Lee cell passaging involves preheating 0.25% trypsin-0.53 mM EDTA digestion solution in a 37°C water bath, aspirating the culture medium from the culture flask, adding 4 mL of PBS to the culture flask, gently shaking and washing, and then discarding it; adding 2 mL of preheated trypsin to the flask, incubating at 37°C for 1 minute, and then adding 2 mL of complete culture medium to terminate the digestion; gently pipetting the cells on the flask wall to completely remove the cells from the flask wall, collecting the cell suspension, centrifuging at 800 rpm for 3 minutes, discarding the supernatant, and resuspending the cells in complete culture medium. The cells were then transferred to a 10 cm culture dish for passaging; 2) After the IOMM-Lee cells reach the passage conditions, rinse the cells three times with PBS, add 1 mL of trypsin, and incubate at 37°C for 1 minute. Rinse the digested cells with complete medium, centrifuge at 800 rpm for 3 minutes, remove the supernatant containing trypsin, FBS, DMEM, and other waste liquids; add 2 mL of PBS, pipette evenly, and centrifuge at 800 rpm for 3 minutes and repeat three times; discard the supernatant after centrifugation, and add appropriate amount of PBS to dilute the cell density to 10 4 / uL; aspirate 10uL of cell suspension for cell counting, and calculate the total number of cells in the centrifuge tube based on the cell count value; aspirate PBS containing a small amount of cell debris and residual DMEM, and then add 5mL PBS to prepare a cell suspension; 3) Construction of IOMM-Lee animal model: Digest RT4 cells in the logarithmic growth phase and resuspend them in 5 mL PBS. Take 10 µL of cells for cell counting and adjust the viable cell concentration to 1 × 10 based on the count results. 4 / uL and 5×10 4 / uL, and establish the IOMM-Lee animal model.

2. The method for establishing a tumor-bearing animal model for an OCT system according to claim 1, wherein: The method of constructing a C6 animal model by culturing a C6 cell line and preparing C6 cells, and constructing a SD rat supratentorial brain parenchymal glioma model, a SD rat optic chiasm glioma model, and a SD rat brainstem glioma model includes: (1) Obtain C6 cells and culture them in a 37°C, 5% CO2 incubator for 24 hours. Change the medium according to the cell growth status and the color change of the culture medium. When the cells cover 80%-90% of the bottom area of ​​the bottle, subculture the C6 cells at a ratio of 1:2 to 1:

3. Continue to culture the subcultured C6 cells in a 37°C, 5% CO2 incubator using high-glucose DMEM medium containing 10% fetal bovine serum. Change the culture medium every 1-2 days according to the growth status, and observe and record the cell growth status every day. (2) After the C6 cells reach the passage conditions, rinse the cells three times with PBS, add 1 mL of trypsin and incubate at 37°C for 1 min; rinse the digested cells with complete medium, centrifuge at 800 rpm for 3 min, remove the trypsin, FBS, DMEM and other waste liquids; add 2 mL of PBS, pipette and blow evenly, centrifuge at 800 rpm for 3 min and repeat three times; discard the supernatant after centrifugation, add appropriate amount of PBS to dilute the cell density to 10 4 / uL; aspirate 10uL of cell suspension for cell counting, and calculate the total number of cells in the centrifuge tube based on the cell count value; aspirate PBS containing a small amount of cell debris and residual DMEM, and then add 5mL PBS to prepare a cell suspension; (3) Digest the C6 cells in the logarithmic growth phase and resuspend them in 5 mL of PBS. Take 10 μL of the cells for cell counting and adjust the viable cell concentration to 1×10 4 / uL, 5×10 4 / uL, 5×10 4 The supratentorial parenchymal glioma model, the chiasmatic glioma model and the brainstem glioma model of SD rats were established respectively.

3. The method for establishing a tumor-bearing animal model for an OCT system according to claim 2, wherein: The C6 cell passaging includes: preheating 0.25% trypsin-0.53 mM EDTA digestion solution in a 37°C water bath, aspirating the culture medium in the culture flask, adding 4 mL of PBS to the culture flask, gently shaking and washing, and then discarding; adding 2 mL of preheated trypsin to the flask, incubating at 37°C for 1 minute, and adding 2 mL of complete culture medium to terminate digestion after digestion; gently blowing the cells on the flask wall with a pipette, collecting the cell suspension after the cells on the flask wall are completely detached, centrifuging at 800 rpm for 3 minutes, discarding the supernatant, adding complete culture medium to resuspend the cells, and transferring the cells to a 10 cm culture dish for passaging.

4. The method for establishing a tumor-bearing animal model for an OCT system according to claim 1, wherein: The SD rat RT4 tumor cell animal model is obtained by culturing the RT4 cell line, preparing the RT4 cells, and establishing the RT4 animal model to construct a rat sciatic nerve sheath tumor model and a rat trigeminal nerve RT4 tumor model, including: 1) Cultivate RT4 cell lines: Obtain RT4 cells and culture them in a 37°C, 5% CO2 incubator for 24 hours. Change the medium based on cell growth and medium color change. When cells have grown to 80%-90% of the bottom area of ​​the flask, passage the RT4 cells at a ratio of 1:2 to 1:

3. Continue culturing the passaged RT4 cells in high-glucose DMEM medium supplemented with 10% fetal bovine serum in a 37°C, 5% CO2 incubator. Change the medium every 1-2 days based on cell growth, and observe and record cell growth daily. 2) Prepare RT4 cells: After RT4 cells reach passage conditions, rinse the cells three times with PBS. Add 1 mL of trypsin and incubate at 37°C for 3 minutes. Rinse the digested cells with complete medium and centrifuge at 1000 rpm for 5 minutes to remove the supernatant containing trypsin, FBS, DMEM, and other waste liquids. Add 2 mL of PBS and pipette evenly. Centrifuge at 1000 rpm for 5 minutes and repeat three times. Discard the supernatant and add PBS to dilute the cells to the desired concentration. Count 10 μL of the cell suspension and calculate the total number of cells in the centrifuge tube based on the cell count. Aspirate the PBS containing a small amount of cell debris and residual DMEM and add 0.5 mL of PBS to prepare the cell suspension. 3) Construction of RT4 animal model: Digest RT4 cells in the logarithmic growth phase and resuspend them in 5 mL of PBS. Take 10 µL of the cells for cell counting and adjust the viable cell concentration to 1 × 10 based on the counting results. 4 / uL and 5×10 4 / uL, and established rat sciatic nerve sheath tumor model and rat trigeminal nerve RT4 tumor model.

5. The method for establishing a tumor-bearing animal model for an OCT system according to claim 4, wherein: The RT4 cell passaging includes: preheating 0.25% trypsin-0.53 mM EDTA digestion solution in a 37°C water bath, aspirating the culture medium in the culture flask, adding 4 mL of PBS to the culture flask, gently shaking and washing, and then discarding; adding 2 mL of preheated trypsin to the flask, incubating at 37°C for 1 minute, and adding 2 mL of complete culture medium to terminate the digestion after digestion; gently blowing the cells on the flask wall with a pipette, collecting the cell suspension after the cells on the flask wall are completely detached, centrifuging at 1000 rpm for 5 minutes, discarding the supernatant, adding complete culture medium to resuspend the cells, and transferring the cells to a 10 cm culture dish for passaging.

6. The method for establishing a tumor-bearing animal model for an OCT system according to claim 1, wherein: The culturing of U87MG cell lines, preparation of U87MG cells, and establishment of a BALB / c nude mouse U87MG tumor cell animal model include: (1) Cultivate U87MG cell line: Obtain U87MG cells and culture them in a 37°C, 5% CO2 incubator for 24 hours. Change the medium according to the cell growth status and the color change of the culture medium. When the cells cover 80%-90% of the bottom area of ​​the bottle, subculture the U87MG cells at a ratio of 1:2 to 1:

3. Continue to culture the subcultured U87MG cells in a 37°C, 5% CO2 incubator using high-glucose DMEM medium containing 10% fetal bovine serum. Change the culture medium every 1-2 days according to the growth status, and observe and record the cell growth status every day. (2) Preparation of U87MG cells: After U87MG cells reach the passage conditions, rinse the cells with PBS three times, add 1 mL of trypsin and incubate at 37°C for 1 min; rinse the digested cells with complete medium, centrifuge at 800 rpm for 3 min, remove the trypsin, FBS, DMEM and other waste liquids; add 2 mL of PBS, pipette and blow evenly, centrifuge at 800 rpm for 3 min and repeat three times; discard the supernatant after centrifugation, add appropriate amount of PBS to dilute the cell density to 10 4 / uL; aspirate 10uL of cell suspension for cell counting, and calculate the total number of cells in the centrifuge tube based on the cell count value; aspirate PBS containing a small amount of cell debris and residual DMEM, and then add 5mL PBS to prepare a cell suspension; (3) Construct the U87MG animal model.

7. The method for establishing a tumor-bearing animal model for an OCT system according to claim 6, wherein: The U87MG cell passaging method includes: preheating a 0.25% trypsin-0.53 mM EDTA digestion solution in a 37°C water bath, removing the culture medium in the culture flask, adding 4 mL of PBS to the culture flask, gently shaking and washing, and then discarding; adding 2 mL of preheated trypsin to the flask, incubating at 37°C for 1 minute, and adding 2 mL of complete culture medium to terminate the digestion after digestion; gently blowing the cells on the flask wall with a pipette, collecting the cell suspension after the cells on the flask wall are completely detached, centrifuging at 800 rpm for 3 minutes, discarding the supernatant, adding complete culture medium to resuspend the cells, and transferring the cells to a 10 cm culture dish for passaging.

8. A tumor-bearing animal model for an OCT system constructed using the method for establishing a tumor-bearing animal model for an OCT system according to any one of claims 1 to 7.

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