Fixture for two-photon recording
By designing a fixator for two-photon recording, and fixing it on the iliac bone and sacrum of mice with bone nails and screws, the problem of anesthesia and suspension in the prior art was solved, and stable calcium imaging recording in the awake and free-acting state of mice was achieved.
Patent Information
- Application Number
- CN202311340131.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-10-16
- Publication Date
- 2025-04-18
AI Technical Summary
The prior art is difficult to stably record the neuronal calcium imaging signals of the dorsal root ganglia of the sacral segment in the state of awakeness and free activity in mice, and the mice need to be anesthetized and suspended during the recording process, resulting in difficulty in recovering the wound and inaccurate research results.
A fixator for two-photon recording is designed, including a body, a mounting hole, a first groove, a first connecting hole and a second connecting hole, which is fixed to the iliac bone and sacrum of the mouse by bone nails and screws. After installation, the mouse can recover and repeat the recording.
Stable imaging of dorsal root ganglion neurons in the mice was achieved in full awake and freely active state, minimizing animal pain and simplifying the implantation and installation process.
Smart Images
Figure CN119837492A_ABST
Abstract
Description
Technical Field
[0001] The present application relates to the technical field of in vivo two-photon calcium imaging, and in particular to a fixture for two-photon recording. Background Art
[0002] Studying neuronal activity in the brain and spinal cord is an important method to reveal neural connections and neuronal functions. In vivo two-photon calcium imaging technology is widely used in central nervous system imaging due to its advantages such as small lens size, stable signal, clear imaging and free activity recording in vivo. The dorsal root ganglion is the first stop for external sensations to enter the central nervous system. It has the functions of identifying, encoding and transmitting peripheral signals. However, due to the lack of a fixture that can stably expose the sacral dorsal root ganglion and support the two-photon lens, the calcium imaging signal of the sacral dorsal root ganglion neurons can only be recorded under anesthesia.
[0003] The method currently used to record the segmental neurons is to clamp the lumbar and coccygeal vertebrae of anesthetized mice using two or more clamps to expose and stabilize the sacral segment in the middle. Although this method can achieve the purpose of recording, it is necessary to cut the entire lumbar and coccygeal back skin and muscle tissue of the mouse to expose the sacral dorsal root ganglia and lumbar and coccygeal vertebrae during the specific implementation process. Due to the dissection and exposure of a large amount of tissue during the experiment, the wound of the mouse after recording is difficult to recover, and repeated recording cannot be achieved. In addition, during the recording process of the above method, the mouse is in anesthesia and suspended state, which may cause differences in the response of neurons to external stimuli, resulting in inaccurate research results.
[0004] It should be noted that the above introduction to the background technology is only for the convenience of providing a clear and complete description of the technical solutions of the present application and facilitating the understanding of those skilled in the art. It cannot be considered that the above technical solutions are well known to those skilled in the art simply because they are described in the background technology section of the present application. Summary of the invention
[0005] The purpose of the present application is to provide a fixture for two-photon recording to solve at least part of the problems pointed out in the above background technology or other similar problems.
[0006] An embodiment of the first aspect of the present application provides a fixator for two-photon recording, comprising: a main body, provided with a mounting hole corresponding to the dorsal root ganglion of the sacral segment of a mouse and used to install a two-photon lens, two first grooves corresponding to the bilateral posterior iliac spines of the mouse and for the bilateral posterior iliac spines to be embedded, two first connecting holes corresponding to the ilium of the mouse, and two second connecting holes corresponding to the sacrum of the mouse; two first connecting members, which are connected to the ilium by being inserted into the two first connecting holes; and two second connecting members, which are connected to the sacrum by being inserted into the two second connecting holes.
[0007] In some embodiments, the body has a lower surface and an upper surface arranged opposite to each other in the thickness direction, a front side surface and a rear side surface arranged opposite to each other in the length direction, and a left side surface and a right side surface arranged opposite to each other in the width direction; the mounting hole extends from the lower surface to the upper surface along the thickness direction; the two first grooves are respectively located on opposite sides of the mounting hole in the width direction, and are both recessed from the lower surface toward the upper surface and extend along the length direction; the two first connecting holes are both arranged adjacent to the front side surface, one of the first connecting holes extends from the left side surface along the width direction and passes through one of the first grooves, and the other first connecting hole extends from the right side surface along the width direction and passes through the other first groove; the two second connecting holes are both arranged adjacent to the rear side surface, and are respectively located between the rear side surface and the two first grooves in the length direction, and the second connecting holes are respectively located on opposite sides of the mounting hole in the width direction, and extend from the lower surface to the upper surface along the thickness direction.
[0008] In some embodiments, the first groove extends from the front side along the length direction to communicate with the mounting hole, and the cross-section of the first groove in the width direction is triangular.
[0009] In some embodiments, the main body is also provided with a first notch, the first notch is located on the front side of the mounting hole and between the two first grooves, the first notch extends from the front side along the length direction to be connected with the mounting hole, and the first notch passes through from the lower surface to the upper surface along the thickness direction.
[0010] In some embodiments, the main body is further provided with a second groove, which is located on the rear side of the mounting hole and between the two second connecting holes, the second groove is recessed from the lower surface toward the upper surface, and extends from the rear side surface along the length direction to be connected with the mounting hole; or, the main body is further provided with a second notch, which is located on the rear side of the mounting hole and between the two second connecting holes, the second notch extends from the lower surface to the upper surface, and extends from the rear side surface along the length direction to be connected with the mounting hole, and the second notch, the mounting hole and the first notch separate the main body into two parts that are not connected to each other.
[0011] In some embodiments, the first connecting member is a steel nail, the second connecting member is a screw, and the second connecting hole is a screw hole.
[0012] The embodiment of the second aspect of the present application provides a fixture for two-photon recording, comprising: a body, having a lower surface and an upper surface arranged oppositely in the thickness direction, a front side surface and a rear side surface arranged oppositely in the length direction, and a left side surface and a right side surface arranged oppositely in the width direction; a mounting hole extending from the lower surface to the upper surface along the thickness direction; two first grooves, respectively located on opposite sides of the mounting hole in the width direction, and both are recessed from the lower surface toward the upper surface and both extend along the length direction; two first connecting holes, both are arranged adjacent to the front side, one of the first connecting holes extends from the left side along the width direction and penetrates one of the first grooves, and the other first connecting hole extends from the right side along the width direction and penetrates the other first groove; two second connecting holes, both are arranged adjacent to the rear side, and are respectively located between the rear side and the two first grooves in the length direction, the second connecting holes are respectively located on opposite sides of the mounting hole in the width direction, and both extend from the lower surface to the upper surface along the thickness direction; two first connecting members, inserted in the two first connecting holes; two second connecting members, inserted in the two second connecting holes.
[0013] In some embodiments, each of the first grooves extends from the front side along the length direction to communicate with the mounting hole, and a cross-section of the first groove in the width direction is a triangle.
[0014] In some embodiments, the main body is also provided with a first notch, the first notch is located on the front side of the mounting hole and between the two first grooves, the first notch extends from the front side along the length direction to be connected with the mounting hole, and the first notch passes through from the lower surface to the upper surface along the thickness direction.
[0015] In some embodiments, the main body is further provided with a second groove, which is located on the rear side of the mounting hole and between the two second connecting holes, the second groove is recessed from the lower surface toward the upper surface, and extends from the rear side surface along the length direction to be connected with the mounting hole; or, the main body is further provided with a second notch, which is located on the rear side of the mounting hole and between the two second connecting holes, the second notch extends from the lower surface to the upper surface, and extends from the rear side surface along the length direction to be connected with the mounting hole, and the second notch, the mounting hole and the first notch separate the main body into two parts that are not connected to each other.
[0016] The beneficial effects of the embodiments of the present application include:
[0017] The fixator for two-photon recording of the present application can be implanted on the ilium and sacrum of mice. During the implantation operation, only the skin and muscles on the surface of the sacrum of the mouse need to be opened. After the fixator is installed, the mouse can be returned to the cage to recover, so that the dorsal root ganglion can be fully exposed while minimizing the pain of the animal, and stable imaging of the dorsal root ganglion neurons in the sacral segment can be achieved when the mouse is fully awake and free to move. The fixator of the present application is fixed on the ilium and sacrum of the mouse by bone nails and screws. The implantation operation is simple and the installation is firm. BRIEF DESCRIPTION OF THE DRAWINGS
[0018] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the prior art descriptions. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work. In the drawings:
[0019] Figure 1 It is a schematic diagram of the three-dimensional structure of a fixture for two-photon recording according to an embodiment of the present application;
[0020] Figure 2 yes Figure 1 A bottom view of a retainer;
[0021] Figure 3 is a schematic diagram of the three-dimensional structure of a fixture for two-photon recording according to another embodiment of the present application;
[0022] Figure 4 is a schematic diagram of a mouse implanted with the holder for two-photon recording of the present application;
[0023] Figure 5 and Figure 6 It is a schematic diagram of a fixator for two-photon recording implanted on the ilium and sacrum of a mouse according to an embodiment of the present application.
[0024] Description of the component numbers in the accompanying drawings:
[0025] 1. body; 101. mounting hole; 102. first groove; 103. first connecting hole; 104. second connecting hole;
[0026] 105, lower surface; 106, upper surface; 107, rear side surface; 108, front side surface; 109, left side surface; 110, right side surface;
[0027] 111, second groove; 112, first notch; 2, first connecting member; 3, second connecting member;
[0028] 100, sacral dorsal root ganglion; 200, two-photon lens; 300, posterior iliac spine; 400, ilium; 500, sacrum. DETAILED DESCRIPTION
[0029] In order to make the purpose, technical solution and advantages of the embodiments of the present application more clear, the embodiments of the present application are further described in detail below in conjunction with the accompanying drawings. Here, the illustrative embodiments of the present application and their descriptions are used to explain the present application, but are not intended to limit the present application.
[0030] In the embodiments of the present application, the terms "first", "second", etc. are used to distinguish different elements in terms of name, but do not represent the spatial arrangement or temporal order of these elements, etc., and these elements should not be limited by these terms. The term "and / or" includes any one and all combinations of one or more of the terms listed in association. The terms "comprising", "including", "having", etc. refer to the presence of the stated features, elements, components or components, but do not exclude the presence or addition of one or more other features, elements, components or components.
[0031] In the embodiments of the present application, the singular forms "a", "the", etc. may include plural forms and should be broadly understood as "a kind" or "a type" rather than being limited to the meaning of "one"; in addition, the term "said" should be understood to include both singular and plural forms, unless the context clearly indicates otherwise; in addition, the term "according to" should be understood as "at least partially based on...", and the term "based on" should be understood as "at least partially based on...", unless the context clearly indicates otherwise; in addition, the term "plurality" means two or more, unless otherwise specified.
[0032] The adjective or adverbial modifiers "upper" and "lower", "front" and "back", "left" and "right" used in this document are used merely to facilitate relative reference between multiple groups of terms and do not describe any specific directional limitation on the modified terms.
[0033] The implementation of the embodiments of the present application will be described below with reference to the accompanying drawings.
[0034] like Figure 1 As shown, an embodiment of the present application provides a fixture for two-photon recording, which includes a main body 1, two first connecting members 2 and two second connecting members 3.
[0035] like Figure 1 , Figure 5 , Figure 6As shown, the body 1 is provided with a mounting hole 101 corresponding to the sacral dorsal root ganglion 100 of the mouse and used to mount the two-photon lens 200, two first grooves 102 corresponding to the bilateral iliac posterior spines 300 of the mouse and for the bilateral iliac posterior spines 300 to be embedded, two first connection holes 103 corresponding to the ilium 400 of the mouse, and two second connection holes 104 corresponding to the sacrum 500 of the mouse. The two first connection members 2 are inserted into the two first connection holes 103 to connect the body 1 to the ilium 400, and the two second connection members 3 are inserted into the two second connection holes 104 to connect the body 1 to the sacrum 500.
[0036] The implantation method of the fixator of the present application is as follows:
[0037] First, the skin, muscles and protruding vertebral spinous processes on the surface of the sacrum 500 of the mouse are surgically opened to expose the sacrum 500 of the mouse;
[0038] Then, the body is placed on the surface of the sacrum 500 of the mouse, so that the two first grooves 102 of the body are stuck in the bilateral iliac posterior spines 300 of the mouse, the two first connecting holes 103 of the body 1 correspond to the ilium 400 of the mouse, and the two second connecting holes 104 of the body 1 correspond to the sacrum 500 of the mouse;
[0039] Subsequently, a first connecting member 2 is inserted into a first connecting hole 103 and the ilium 400, and another first connecting member 2 is inserted into another first connecting hole 103 and the ilium 400, so as to connect the body 1 with the ilium 400; and a second connecting member 3 is inserted into a second connecting hole 104 and the sacrum 500, and another second connecting member 3 is inserted into another second connecting hole 104 and the sacrum 500, so as to connect the body 1 with the sacrum 500;
[0040] Afterwards, the two-photon lens 200 is mounted on the mounting hole 101 to record the neuronal calcium signal of the sacral dorsal root ganglion 100 when the mouse is awake and moves freely.
[0041] exist Figure 1 and Figure 2 In the example, the body 1 has a lower surface 105 and an upper surface 106 arranged opposite to each other in its thickness direction H, a front side surface 108 and a rear side surface 107 arranged opposite to each other in its length direction L, and a left side surface 109 and a right side surface 110 arranged opposite to each other in its width direction W.
[0042] like Figure 1 As shown, the mounting hole 101 extends from the lower surface 105 to the upper surface 106 along the thickness direction H. In other words, the mounting hole 101 penetrates the body 1 along the thickness direction H to form a through hole.
[0043] like Figure 1As shown, the two first grooves 102 are respectively located on opposite sides of the mounting hole 101 in the width direction W, and are both recessed from the lower surface 105 toward the upper surface 106 and extend along the length direction L. Specifically, the first groove 102 extends a preset length from the front side 108 toward the rear side 107, and the front end of the first groove 102 passes through the front side 108 of the main body 1, and the rear end of the first groove 102 is connected to the mounting hole 101, so that the first groove 102 can clamp the bilateral iliac posterior spines 300, or in other words, it is convenient for the bilateral iliac posterior spines 300 to be embedded in the first groove 102, so as to prevent the main body 1 from moving left and right relative to the sacral dorsal root ganglion 100 of the mouse.
[0044] like Figure 1 As shown, the cross-section of the first groove 102 in the width direction W is triangular in shape to match the shape of the posterior iliac spine 300 .
[0045] like Figure 1 As shown, the two first connection holes 103 are both arranged adjacent to the front side 108, the first connection hole 103 on the left side extends from the left side 109 along the width direction W and passes through the first groove 102 on the left side, and the first connection hole 103 on the right side extends from the right side 110 along the width direction W and passes through the first groove 102 on the right side. Therefore, the first connection member 2 on the left side can be inserted into the first connection hole 103 on the left side from the left side 109 of the body 1 and pass through the posterior iliac spine 300 in the first groove 102 on the left side, and the first connection member 2 on the right side can be inserted into the first connection hole 103 on the right side from the right side 110 of the body 1 and pass through the posterior iliac spine 300 in the first groove 102 on the right side, thereby connecting the body 1 to the ilium 400, preventing the body 1 from moving forward and backward or up and down relative to the sacral dorsal root ganglion 100 of the mouse.
[0046] like Figure 1 As shown, the two second connection holes 104 are both arranged adjacent to the rear side surface 107, and are respectively located between the rear side surface 107 and the two first grooves 102 in the length direction L, and are respectively located on opposite sides of the mounting hole 101 in the width direction W, and each second connection hole 104 extends from the lower surface 105 to the upper surface 106 along the thickness direction H. Therefore, the two second connection members 3 can respectively pass through the two second connection holes 104 and be inserted into the sacrum 500, thereby connecting the body 1 to the sacrum 500, and preventing the body 1 from moving forward and backward or left and right relative to the sacral dorsal root ganglion 100 of the mouse.
[0047] As Figure 1As shown, the body 1 is further provided with a first notch 112 located at the front side of the mounting hole 101 and between the two first grooves 102. The first notch 112 extends from the front side 108 along the length direction L to communicate with the mounting hole 101, and the first notch 112 penetrates from the lower surface 105 to the upper surface 106 along the thickness direction H; the second groove 111 is located at the rear side of the mounting hole 101 and between the two second connection holes 104. The second groove 111 is recessed from the lower surface 105 toward the upper surface 106, and extends from the rear side 107 along the length direction L to communicate with the mounting hole 101. Therefore, the first notch 112 and the mounting hole 101 together separate the body 1 into a left part and a right part connected only by the second groove 111 to avoid the mouse vertebrae protruding in the middle.
[0048] However, the present application is not limited thereto. In another example, Figure 3 As shown, the second groove 111 can be replaced by a second notch 113. The difference between the second notch 113 and the second groove 111 is that the second notch 113 extends from the lower surface 105 to the upper surface 106. Therefore, the second notch 113, the first notch 112 and the mounting hole 101 together separate the main body 1 into a left part and a right part that are not connected to each other. The two parts can be detachably connected by an additional connecting member (such as a bolt), and the middle spine of the mouse can also be avoided. In addition, the distance between the two parts can be adjusted to accommodate mice of different sizes.
[0049] exist Figure 1 In the example, the first connecting member 2 is a steel nail (ie, a bone nail), the second connecting member 3 is a screw, and the second connecting hole 104 is a screw hole.
[0050] Optionally, the bone nail may be a stainless steel nail with a length of 4 mm, and the screw may be a stainless steel screw with a length of 3 mm.
[0051] Optionally, the body 1 may be made of stainless steel.
[0052] When making the fixator of the present application, a digital model of the mouse pelvis can be established through anatomical measurement and computer simulation, and then the fixator can be made by combining data modeling and CNC precision processing technology.
[0053] When using the existing clamp, it is necessary to cut open the entire lumbar and sacral dorsal skin and muscle tissue to expose the sacral dorsal root ganglia and lumbar and coccygeal vertebrae. However, when using the fixator of the present application, it is only necessary to open the skin and muscles on the surface of the mouse's sacrum. After the fixator is installed, the mouse can be returned to the cage to recover, thereby fully exposing the dorsal root ganglia while minimizing the animal's pain. The mouse suffers less trauma and recovers faster.
[0054] The fixator of the present application is fixed on the ilium and sacrum of the mouse by bone nails and screws, and the implantation operation is simple and the installation is firm.
[0055] like Figure 4 As shown, mice equipped with a fixture and a two-photon lens can repeatedly record calcium signals from neurons in the sacral dorsal root ganglia and achieve stable imaging of neurons in the sacral dorsal root ganglia. Since the mice are fully awake and free to move during the recording process, the calcium signals of neurons can be accurately recorded in real time.
[0056] The specific embodiments described above further illustrate the purpose, technical solutions and beneficial effects of the present application. It should be understood that the above description is only a specific embodiment of the present application and is not intended to limit the scope of protection of the present application. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present application should be included in the scope of protection of the present application.
Claims
1. A holder for two-photon recording, comprising: The body is provided with a mounting hole corresponding to the dorsal root ganglion of the sacral segment of the mouse and used to mount the two-photon lens, two first grooves corresponding to the bilateral iliac posterior spines of the mouse and for the bilateral iliac posterior spines to be embedded, two first connection holes corresponding to the ilium of the mouse, and two second connection holes corresponding to the sacrum of the mouse; Two first connecting members, connected to the body and the ilium by being inserted into the two first connecting holes; Two second connecting members are inserted into the two second connecting holes to connect the body to the sacrum.
2. The fastener according to claim 1, wherein: The body has a lower surface and an upper surface arranged oppositely in the thickness direction thereof, a front side surface and a rear side surface arranged oppositely in the length direction thereof, and a left side surface and a right side surface arranged oppositely in the width direction thereof; The mounting hole extends from the lower surface to the upper surface along the thickness direction; The two first grooves are respectively located at opposite sides of the mounting hole in the width direction, and are both recessed from the lower surface toward the upper surface and extend along the length direction; Two first connection holes are both arranged adjacent to the front side surface, wherein one of the first connection holes extends from the left side surface along the width direction and passes through one of the first grooves, and the other first connection hole extends from the right side surface along the width direction and passes through the other first groove; The two second connection holes are both arranged adjacent to the rear side surface and are respectively located between the rear side surface and the two first grooves in the length direction. The two second connection holes are respectively located on opposite sides of the mounting hole in the width direction and extend from the lower surface to the upper surface along the thickness direction.
3. The fastener according to claim 2, wherein: The first groove extends from the front side surface along the length direction to communicate with the mounting hole, and the cross-section of the first groove in the width direction is a triangle.
4. The fastener according to claim 2, wherein: The main body is also provided with a first notch, which is located on the front side of the mounting hole and between the two first grooves. The first notch extends from the front side along the length direction to communicate with the mounting hole, and the first notch passes through from the lower surface to the upper surface along the thickness direction.
5. The fastener according to claim 4, wherein: The body is further provided with a second groove, the second groove is located at the rear side of the mounting hole and between the two second connection holes, the second groove is recessed from the lower surface toward the upper surface, and extends from the rear side surface along the length direction to communicate with the mounting hole; or, The body is also provided with a second notch, which is located at the rear side of the mounting hole and between the two second connecting holes. The second notch extends from the lower surface to the upper surface and extends from the rear side surface along the length direction to communicate with the mounting hole. The second notch, the mounting hole and the first notch separate the body into two parts that are not connected to each other.
6. The fastener according to claim 1, wherein: The first connecting member is a steel nail, the second connecting member is a screw, and the second connecting hole is a screw hole.
7. A holder for two-photon recording, wherein: include: The body has a lower surface and an upper surface arranged opposite to each other in the thickness direction, a front side surface and a rear side surface arranged opposite to each other in the length direction, and a left side surface and a right side surface arranged opposite to each other in the width direction; A mounting hole extending from the lower surface to the upper surface along the thickness direction; Two first grooves are respectively located at opposite sides of the mounting hole in the width direction, are both recessed from the lower surface toward the upper surface and extend along the length direction; Two first connection holes, both disposed adjacent to the front side surface, wherein one of the first connection holes extends from the left side surface along the width direction and passes through one of the first grooves, and the other first connection hole extends from the right side surface along the width direction and passes through the other first groove; Two second connection holes are arranged adjacent to the rear side surface and are respectively located between the rear side surface and the two first grooves in the length direction, and the second connection holes are respectively located on opposite sides of the mounting hole in the width direction and extend from the lower surface to the upper surface along the thickness direction; Two first connecting members, inserted into the two first connecting holes; Two second connecting members are inserted into the two second connecting holes.
8. The fastener according to claim 7, wherein: Each of the first grooves extends from the front side surface along the length direction to communicate with the mounting hole, and the cross-section of the first groove in the width direction is a triangle.
9. The fastener according to claim 7, wherein: The main body is also provided with a first notch, which is located on the front side of the mounting hole and between the two first grooves. The first notch extends from the front side along the length direction to communicate with the mounting hole, and the first notch passes through from the lower surface to the upper surface along the thickness direction.
10. The fastener according to claim 9, wherein: The body is further provided with a second groove, the second groove is located at the rear side of the mounting hole and between the two second connection holes, the second groove is recessed from the lower surface toward the upper surface, and extends from the rear side surface along the length direction to communicate with the mounting hole; or, The body is also provided with a second notch, which is located at the rear side of the mounting hole and between the two second connecting holes. The second notch extends from the lower surface to the upper surface and extends from the rear side surface along the length direction to communicate with the mounting hole. The second notch, the mounting hole and the first notch separate the body into two parts that are not connected to each other.