WO2016072873A1

WO2016072873A1 - Device and assembly for immobilizing an animal, use of such device and method for immobilizing an animal

Info

Publication number: WO2016072873A1
Application number: PCT/PL2015/050058
Authority: WO
Inventors: Kacper LUKASIEWICZ; Magdalena ROBACHA; Marzena STEFANIUK; Artur WOLNY; Katarzyna RADWAŃSKA
Original assignee: Instytut Biologii Doswiadczalnej Im. M. Nenckiego Polskiej Akademii Nauk
Priority date: 2014-11-04
Filing date: 2015-11-04
Publication date: 2016-05-12
Also published as: PL410001A1; PL224680B1

Abstract

A device (1) for immobilizing an animal in order to examine a selected area of the animal's body, the device (1) comprising two upright members (2a, 2b) secured by their lower ends to a base (3), the height of the upright members (2a, 2b) being adjustable independently of one another and being adapted to be fixed at a chosen position, the device further comprising a retention bar (4) having two ends (4a, 4b), each end (4a, 4b) being engageable with an upper end of one upright member (2a, 2b) by means of a connection structure (7a, 7b) enabling the retention bar (4) to be rotated around its longitudinal axis (X) and to be fixed in a chosen rotated position, an oblong support element (9) being releasably fixed to the retention bar (4) between its two ends (4a, 4b), the support element (9) having a proximal end (9a) adapted to be releasably fixed to the retention bar (4) and a distal end (9b) adapted to be releasably fixed to the animal body. A use of the device and an assembly comprising the device according to the invention and a number of exchangeable support elements. A method of immobilizing an animal.

Description

Device and assembly for immobilizing an animal, use of such device and method for immobilizing an animal

[0001] The present invention relates to a device, an assembly and a method for immobilizing an animal, in particular in the field of animal restraint and immobilisation for the purposes of scientific and surgical procedures, particularly those performed under a microscope, particularly 2-photon in vivo imaging.

[0002] Animal immobilisation is a necessary procedure for performing a great many procedures in biological science research as well as in veterinary medicine. These procedures, particularly those performed under large magnifications, e.g. by means of microscopes, require fine positioning of the animal. The immobilizing means or devices used in such a procedure should fulfill some basic conditions. Firstly, it should safely immobilise the animal preventing it from shifting in any way during the procedure, without any harm coming to the animal. Secondly, it should position the animal or a part thereof in an optimal way for performing the procedure. Thirdly, the means or device cannot interfere with other means, devices or actions used in the actual or preparatory procedure. Additionally, the immobilising means or device should be scalable, to optimally match its size to the animal on which a given procedure is performed.

[0003] Areas of research with a particular need for effective, safe and repeatable animal immobilisation include in vivo observation of biological processes, cell collection and microsurgery in research animals performed with the aid of microscopes, particularly epi-fluorescent and confocal microscopes. One such particular area is the in vivo observation of fluorescent or fluorescent-tagged cells in animal brains through a transcranial window using a 2-photon microscope.

[0004] 2-photon microscopy is a novel and revolutionary approach into in vivo brain imaging. It enables the visualization of morphological and functional changes deep in the brain tissue of living animals through a so-called "cranial window": a hole in the cranium covered with a cover glass. The animal can be put under the microscope in either a conscious state or under anaesthesia. An increasing number of laboratories use 2-photon microscopes, implementing the technique throughout the scientific world as a new standard for in vivo brain imaging. While the technique is quickly gaining popularity, the physical set-ups and methodologies for 2-photon microscopy are still very much under development.

[0005] One of the most challenging parts of obtaining the data from a 2-photon in vivo imaging experiment proves to be the correct placement of the animal under the microscope. It is crucial that the head of the animal is immobilized. Even unavoidable breathing movements significantly impede the subsequent analysis process. Reduction of the head movement to a necessary minimum is a fundamental way of greatly improving the quality of the obtained images. Another challenge is that most of the in vivo experiments have a long duration, featuring multiple viewing sessions per animal. This requires a means to affix the animal under the microscope in a reproducible, quick and consistent way.

[0006] Commercially available microscopy setups do not provide satisfactory means to place and hold the animal under the microscope and consequently installation of additional holders is necessary. Although there are many experimental designs, an all-purpose immobilizing device is crucially needed. Such a device would allow a researcher to standardize the procedures so as to perform highly reproducible experiments, from bench to bench, and between laboratories.

[0007] At present, there is only one commercially available immobilisation device or holder applicable for 2-photon microscopy and in vivo imaging. Most laboratories use a wide variety of custom made or in-house built holders which are not standardized in any way and present a series of distinct disadvantages.

[0008] There are two most popular devices of the above described type, namely a standard stereotaxic frame and a commercially available holder by Luigs & Neumann, both having a similar structure and principle of operation.

[0009] The Luigs & Neumann holder is a "U" shaped frame movably supported on three ball bearings, each arm of the "U" being equipped with 4 sets of bars for cranium immobilization. The bars are adjusted by screws. The problem with this solution is that it is impossible to assess the force applied on the animal's cranium, which often proves lethal for the animal. The device does not provide any means of marking the correct settings for the animal, so each time it is immobilised, the position slightly differs. It might be a challenge when it comes to long-term experiments, when it is essential to have comparable data acquired at different time points. It is also impossible to use in experiments on conscious animals.

[0010] The standard stereotaxic frame is used in some laboratories during imaging sessions. It poses the same difficulties as the previously described commercial holder. Although the stereotaxic frame is more safe in usage than the commercial holder, because of the more controllable way of adjusting the ear bars, its application for in vivo imaging is still greatly limited. Adaptors for stereotaxic frame applied for in vivo imaging can be obtained commercially but they do not eliminate the problems connected with the use of the standard frame. An additional difficulty is the common obstruction of access for the microscope objective, and thus suboptimal imaging angles.

[0011] Other setups include attaching a large bar or plate to the head of the animal either permanently or only for the time of visualization session. The bar is then attached to the fixed stage, allowing a relatively strong and movement-free set for the animal. The first solution, however, poses a great inconvenience for the animal between the experimental sessions and limits the possible behavioral treatments to those without any spatial restrictions. The second requires operating the animal before every imaging session to attach and the disassemble the bar, which is both laborious and poses great danger of infection, as well as possibly affecting the outcome of the experiment.

[0012] Smaller bars and other designs are also used in similar fashion. The bar is permanently glued into the cranium to be later attached to a fixed stand. Although on a smaller scale, these solutions also cause problems of spatial restriction. In an environment such as an IntelliCage, the animal is required to enter a small round door to get to the bottle of water. The metal bar attached to the head might cause trouble getting into the door or batter on the roof of a small space, compromising the cranial window that is placed on the same dental cement "cap" as the bar.

[0013] The aim of the present invention was to solve the above stated problems by providing a device, an assembly and a method for immobilizing an animal enabling faster, more convenient and more precise immobilization of the animal. [0014] A particular aim of the invention was to facilitate adjustment of the level and the angle of positioning the animal in either a conscious or an anaesthetized state and to reduce the time required to prepare the animal for imaging by several-fold.

[0015] Another aim of the invention was to provide a repeatedly adjustable and scalable immobilization device that could be used with an animal having a permanent working site, such as e.g. a cranial window, placed on the body.

[0016] An additional aim was to provide a standardized device of a simple construction in order to make it adaptable for a wide range of uses, e.g. for different parts of different animal species.

[0017] The device has the benefit of solving two difficult problems which bother life science researchers: firstly, the use of a standardized immobilising device is convenient for multiple users and sites; secondly, the standardized immobilising device imparts the standardization of experimental design. The present disclosure is of a particular significance to the scientific research on the living animal brain, particularly the research using imaging techniques.

[0018] According to the invention a device is provided for immobilizing an animal in order to examine a selected area of the animal's body, the device comprising two upright members secured by their lower ends to a base, the height of the upright members being adjustable independently of one another and being adapted to be fixed at a chosen position, the device further comprising a retention bar having two ends, each end being releasably engageable with an upper end of one upright member by means of a connection structure enabling the retention bar to be rotated around its longitudinal axis and to be fixed in a chosen rotated position, an oblong support element being releasably fixed to the retention bar between its two ends, the support element having a proximal end adapted to be releasably fixed to the retention bar and a distal end adapted to be releasably fixed to the animal body.

[0019] The support element preferably extends transversally to the retention bar and has a longitudinal axis as well as two engagement surfaces, one at the proximal end and the other at the distal end, the engagement surfaces being substantially parallel to the axis and lying in the planes that form an angle ranging from 0° to 90° between each other. [0020] Each upright member is preferably formed by a telescoping pole composed of at least two substantially concentric elements.

[0021] The telescoping poles may be fixed at a chosen vertical position by locking bolts, preferably thumbscrews.

[0022] Preferably, each connection structure is formed by a hollow sleeve connected transversally to the upright member, each end of the retention bar being located within the hollow sleeve and being held in the chosen rotated position by means of a locking bolt, preferably a thumbscrew extending through the wall of the hollow sleeve.

[0023] Each hollow sleeve preferably has an internal diameter adapted to accommodate the end of the retention bar with a play so that the heights of the upright members may be different.

Preferably, the lower ends of the upright members are integral with the base.

[0024] The support element preferably is a plate-like element having a helical twist about its centre, its engagement surfaces being constituted by its regions near the ends.

[0025] In another embodiment, the support element is a bar having a central part of a circular cross-section and two bilaterally recessed parts at the ends comprising the flat engagement surfaces.

[0026] The support element may be affixed to the retention bar by means of a locking bolt, preferably a thumbscrew.

[0027] Preferably, the angle between the planes in which the engagement surfaces lie ranges from 20° to 35°.

[0028] Preferably, said angle is selected to match the position of the part of the animal body that it is fixed to the support element and to the area of its body to be examined.

[0029] The engagement surfaces of the support element may be provided with apertures for receiving fixing means, preferably the apertures are threaded and the fixing means are thumbscrews.

[0030] The device may be sized to match the animal being worked on, particularly preferably the size of laboratory rodents. [0031] Preferably, the components of the device are made of a material that is rigid enough to restrain the animal in position, preferably stainless steel or high-grade aluminum.

[0032] The height of each upright member is preferably scaled in order to enable repeatable positioning of the animal.

[0033] Preferably, the rotation of the retention bar around its longitudinal axis is scaled in order to enable repeatable positioning of the animal.

[0034] According to another aspect of the invention, an assembly is provided for immobilizing an animal in order to examine a selected area of the animal's body, the assembly comprising the device according to the invention and a number of exchangeable support elements, each support element having its engagement surfaces defining a different angle therebetween.

[0035] According to yet another aspect of the invention, use of the device according to the invention is provided, for immobilisation of an animal during a surgical or scientific procedure.

[0036] Preferably, the animal is anaesthetized during the surgical or scientific procedure.

[0037] Also preferably, the procedure is a procedure performed under a microscope such as a stereoscope, a light microscope or a fluorescent microscope, preferably a confocal microscope, most preferably a 2P microscope, or any other microscope capable of imaging in vivo.

[0038] The procedure may be performed on the animal's head, preferably on its brain, particularly preferably through a trans-cranial window.

[0039] A method according to the invention, of immobilising an animal by means of the device according to the invention, in order to examine a selected area of the animal's its body, the animal having a fixing member (14) attached close to said selected part of its body, comprises the following steps:

[0040] connecting the engagement surface at the distal end of the support element to the fixing element attached to the animal's its body;

[0041] connecting the engagement surface at the proximal end of the support element to the retention bar; [0042] inserting the ends of the retention bar in to the respective connection structures;

[0043] adjusting and fixing the height of each upright member as well as the rotation angle of the retention bar to match the size and shape of the animal, so that the selected part of the animal's body is in a position enabling examination.

[0044] In another aspect the invention provides a method of immobilising an animal by means of the assembly according to the invention, in order to examine a selected area of the animal's body, the animal having a fixing member attached close to said selected part of its body, comprises the following steps:

• selecting a support element having a suitable angle and connecting it to the retention bar;

• connecting the engagement surface at the distal end of the support element to the fixing element attached to the animal's its body;

• connecting the engagement surface at the proximal end of the support element to the retention bar;

• inserting the ends of the retention bar in to the respective connection structures;

• adjusting and fixing the height of each upright member as well as the rotation angle of the retention bar to match the size and shape of the animal, so that the selected part of the animal's body is in a position enabling examination.

[0045] Preferably, the method further comprises exchanging at least once the selected support element with another support element having a different angle in order to improve the positioning of the selected part of the animal's body.

[0046] The selected part of the animal's body is preferably positioned in a way allowing access to a transcranial window for performing procedures on an animal's brain, preferably under a microscope, preferably for imaging using a 2P microscope.

[0047] Exemplary embodiments of the present invention are illustrated in the attached drawing, its figures being by no means limitative to the scope of the invention. A person skilled in the art will be able to produce multiple other embodiments falling within the scope of the present invention. The drawing comprises the following figures:

Figure la shows a perspective view of the device according to the invention set in a first exemplary position;

Figure lb shows a top view of the device shown in fig. la;

Figure lc shows a front view of the device shown in fig. la;

Figure Id shows a side view of the device shown in fig. la;

Figure 2a shows a perspective view of the device according to the invention set in a second exemplary position ;

Figure 2b shows a top view of the device shown in fig. 2a;

Figure 2c shows a front view of the device shown in fig. 2a;

Figure 2d shows a side view of the device shown in fig. 2a;

Figures 3a and 3b show respectively a side view and a perspective view of the upper concentric element of the upright member in an enlarged scale;

Figures 4a and 4b show respectively a side view and a perspective view of the lower concentric element of the upright member in an enlarged scale;

Figures 5a_; 5b and 5c show respectively a top view a perspective view and a front view of a first embodiment of the support element in an enlarged scale;

Figures 6a and 6b show respectively a top view and a perspective view of a second embodiment of the support element in an enlarged scale;

Figures 7a and 7b show respectively a top view and a perspective view of a third embodiment of the support element in an enlarged scale;

Figures 8a and 8b show a top view and a perspective view of the retention bar;

Figure 9 schematically shows a perspective view of the device according to the invention with an animal attached thereto.

[0048] As may be seen in figs, la, lb, lc, Id, the immobilising device 1 according to the present invention comprises two upright members 2a and 2b secured by their lower ends to a base 3 and a retention bar 4 arranged substantially at the upper ends of the two upright members 2a and 2b. The height of each upright member may be adjusted independently on one another, so that the retention bar 4 may be positioned horizontally or not horizontally. In the shown embodiment each upright member 2a, 2b is a telescopic pole composed of two substantially concentric elements, the inner (e.g. upper) element 5 and an outer (e.g. lower) element 5'. The telescoping poles may be fixed at a chosen vertical position by locking bolts 6a, 6b, preferably thumbscrews. The concentric elements 5 and 5' are shown in an enlarged scale in fig. 3a, 3b and 4a, 4b. The lower ends of the upright members 2a, 2b may be fixable or may be integral with the base 3. A man skilled in the art will be able to choose other forms of conventional upright members of adjustable heights that may be fixed in a chosen vertical position which would also be encompassed by the present invention.

[0049] Each upright member 2a, 2b, has a connection structure 7a, 7b fixedly attached on its top to enable the retention bar 4 to be installed. The connection structure 7a, 7b may be integral with the upright members 2a, 2b.

[0050] In the described embodiment, the connection structures 7a, 7b have a form of hollow sleeves connected transversally to one end of each upper element of the upright members 2a, 2b. A man skilled in the art will appreciate that the connection structures 7a, 7b may also have different forms as long as they enable the retention bar 4 to be releasably installed or engaged by its two ends 4a, 4b at the ends of the upright members 2a, 2b so that the retention bar 4 may be rotated around its longitudinal axis X and to be fixed in a chosen rotated position. Here, locking bolts 8a, 8b, each consisting of a thumbscrew extending through the wall of the hollow sleeve 7a, 7b, are provided for fixing the retention bar 4 in the chosen angular position but other suitable means may also be used. In figs, la -Id the retention bar 4 is installed horizontally, i.e. the upright members 2a and 2b have the same height. Additionally, the locking bolts 8a, 8b fix the retention bar 4 against axial movement.

[0051] As may be best seen in figs, la and lc, an oblong support element 9 is releasably fixed to the retention bar 4 between its two ends 4a, 4b. The support element 9 has a proximal end 9a and a distal end 9b. Preferably, the support element 9 extends transversally to the retention bar 4. The function of the support element 9 is to enable to fix and immobilize the animal in the device 1 according to the invention. Therefore, the support element 9 has its proximal end adapted to be releasably fixed to the retention bar 4 and its distal end 9b adapted to be releasably fixed to the animal's body. The proximal end 9a is fixed to the retention bar 4 preferably by means of a fixing bolt 10, e.g. a thumbscrew. The distal end 9b is fixed to the animal's body by similar conventional means, e.g. a thumbscrew 11 and nut assembly, the nut 14 being fixed to the animal's body. Three exemplary shapes of the support element 9 will be further described in detail in reference to figs. 5a-5b, 6a-6b, 7a-7b.

[0052] In figs. 2a -2d the same exemplary embodiment of the device 1 is shown with the retention bar 4 installed in a slanted position, i.e. the upright members 2a and 2b have different heights. The choice of the angle of inclination of the retention bar 4 is possible due to the fact that the connection structures 7a, 7b, in the described embodiment the hollow sleeves, have their internal diameters adapted to accommodate the ends 4a and 4b of the retention bar 4 with a play. The respective sizes and diameters of the retention bar, the upright members and the connection structures as well as the play may easily be chosen by a man skilled in the art depending on the actual requirements (e.g. the size of the examined animal).

[0053] As mentioned above, the support element 9 has a generally oblong shape with a proximal end 9a adapted and a distal end 9b. Three exemplary shapes of the support element 9 are shown in detail in figs. 5a-5b, 6a-6b, 7a-7b.

[0054] Figs. 3a and 3b show the inner upper element 5 of the upright member 2a, 2b in a side view and in a perspective view respectively. In this embodiment the element 5 is tubular with a transversal tubular hollow sleeve 7a, 7b at one end and it is provided with a scale enabling fixing the chosen height of the upright member 2a, 2b. The scale may be realized in any suitable conventional form, e.g. a series of protrusions, grooves or holes etc. cooperating with the locking bolts 6a, 6b inserted into fixing apertures of the outer lower element 5'.

[0055] Figs. 4a and 4b show the outer lower element 5' in a side view and in a perspective view respectively having the fixing apertures.

[0056] Fig. 5a, 5b and 5c present the support element 9 having a longitudinal axis Y as well as two engagement surfaces, one at the proximal end 9a and the other at the distal end 9b. The engagement surfaces are formed at or are constituted by the end regions 9a, 9b of the support element 9 and they are substantially parallel to the axis Y. Besides, the engagement surfaces lie in the planes that form an angle a ranging from 0° to 90° between each other. Fig. 5c, being an even more enlarged front view of the support element 9, the angle a is schematically shown. In the embodiments shown in figs. 5a, 5b and 5c the support element 9 is a plate-like element having a helical twist about its centre and the angle a is about 35°. The ends 9a and 9b are provided with apertures 12a and 12b for receiving fixing means, preferably the apertures 12a and 12b are threaded and the fixing means are thumbscrews.

[0057] Figs. 6a and 6b present the support element 9 in its simplest form, i.e. a flat rectangular plate with a fixing aperture at each end. The angle a is 35°. Such a support element 9 may be used if it is possible to arrange the animal's body in the required positon by adjusting the slant of the retention bar 4 only by setting the upright members 2a and 2b at suitable heights and by adjusting the angle of rotation of the retention bar 4. However, the scope of the adjustment of the slant of to the retention bar 4 is limited, in particular by its diameter and the internal diameter of the hollow sleeves 7a, 7b. In some situations it is necessary to further adjust the angle at which the animal's body is fixed to the device 1. Therefore, a set of exchangeable support elements 9, each having its engagement surfaces defining a different angle a therebetween may be provided with the device according to the invention, in order to enable a ready possibility to further adjust the animal's position. The device 1 and the set of the exchangeable support elements 9 constitute the assembly according to the invention.

[0058] Another embodiment of the support element 9 is shown in figs. 7a, 7b and 7c in which it is a bar having a central part of a circular cross-section and two bilaterally recessed parts at the ends 9a, 9b comprising the flat engagement surfaces. In this embodiment the angle a may also range from 0° to 90°.

[0059] In figs. 8a and 8b an embodiment of the retention bar 4 is shown in a top and perspective views. As may be seen, the retention bar 4 as shown has a circular cross- section and an engagement recessed region 4c between its ends 4a, 4b, the shape of the recessed region 4c being adapted to be connected with the proximal end 9a of the support member 9. Both ends 4a, 4b are provided with a scale for adjustment of the angle of rotation of the retention bar 4. The scale may be realized in any suitable conventional form, e.g. a series of protrusions, grooves or holes etc. cooperating with the locking bolts 8a, 8b inserted into fixing apertures of the hollow sleeves 7a, 7b. [0060] The recessed region 4c has an aperture, preferably a threaded aperture, for receiving the fixing bolt 10.

[0061] The device 1 according to the invention may obviously be sized to match the animal being worked on, particularly preferably the size of laboratory rodents. Its main components, i.e. the upright members, the connection structures, the support elements are preferably made of a material that is rigid enough to restrain the animal in position, preferably stainless steel or high-grade aluminum. Other materials meeting the above requirements may also be envisages.

[0062] In fig. 9 a perspective view of an example of the device 1 according to the invention with an animal attached thereto is presented. With reference to fig. 9, an example of the use of the described preferable embodiment of the immobilisation device according to the present invention in 2-photon microscopy of the brain will be described.

[0063] During a cranial window 13 implantation operation a fixing member 14, e.g. a nut of an appropriate size is affixed by known means to the scull of the animal. It is later attached to the distal end 9b of the support element 9 by means of a thumbscrew passing through the aperture 12b, the thumbscrew thread being compatible with the nut 14 on the animal's head. Subsequently, the other aperture 12a in the opposite proximal end 9a of the support element 9 is attached to the retention bar by means of the locking bolt 10 such as a thumbscrew. In fig. 9 a flat plate support element 9 is shown, but provided that there is a number of different support elements 9 available with different angles a, another support element 9 with a helical twist may be matched to the shape of the animal worked on depending on the needs of the procedure. The support elements 9 included in the assembly according to the invention may be exchanged during the process of immobilization in order to improve the position of the animal.

[0064] The retention bar 4 is then inserted into the connection structures 7a and 7b and the height of the adjustable upright members 2a and 2b and thus the transverse angle (slant angle) of the retention bar 4 is selected and fixed to a pre-set value either empirically, or else using the graduation or scales on the inner members 5. The extension (height) of each upright member 2a, 2b is locked using the locking bolts 6a, 6b and finally the optimal rotational angle of the retention bar 4 is adjusted and locked within the connection structures 7a and 7b using the locking bolts 8a, 8b. In fig. 9 the retention bar 4 is horizontal.

[0065] As the upright members 2a, 2b and the retention bar 4 feature scale marks, and each support element 9 is marked so that its twist angle may be easily identified, the correct placement of the examined animal may be easily reproduced for the same animal at different time points of an experiment.

[0066] If needed, the animal's head can be additionally supported using pads, cushions, shaped seats or shims, wedges or any other additional aids designated 15 in fig. 9, used individually or combined and either placed loosely or attached to the base 3 or device 1 so as to prop the animal body part in a desirable position.

[0067] The quality of the acquired data greatly depends on the correct placement of the cranial window 13 in relation to the lens of the microscope. The place should be reproducible in different time points. The best way to visualize the brain tissue in repetitive way is to place the cranial window 13 in parallel with the lens, and to keep exactly the same position during different visualization sessions. The device according to the present invention facilitates the adjustment of the animal's head in all axes, measurement and recording of the optimal position for a given animal, providing a means to quickly obtain the perfect set-up for said animal in multiple instances.

[0068] The device according to the invention can be easily adjusted to any 2-photon microscope system, either commercially available or custom made. With certain modifications depending on the experimental design, it can also be applied for experiments with conscious and active animals which require more sophisticated tables, runways or arenas. The adjustable height of the upright members allows easy placement of a pad, heating blanket or a moving construction such as the catwalk underneath the animal.

[0069] The immobilisation device is highly adjustable. Possible modifications include implanting a guide, such as a tongue and groove shape or cam, inside the columns to avoid the rotation of the vertical members. The design of the upper concentric element versus the lower concentric element movement may be improved by applying a thread inside the upper concentric element and a screw inside the lower concentric element, so that the movement may be controlled by rotation of the screw. If necessary, the whole process might be mechanized for an easier setting of the right position of the animal and more standardization. This can be easily achieved by installing small engines connected with every moving part of the device. The device itself may include sealed thread holes, compatible with the standard microscopy table mounting holes.

[0070] The immobilisation device according to the invention allows an easy adjustment of the device on a microscopy table and also mounting of additional accessories, for example drinkers, feeders, levers etc. the procedure is a procedure performed under a microscope such as a stereoscope, a light microscope or a fluorescent microscope, preferably a confocal microscope, most preferably a 2P microscope, or any other microscope capable of imaging in vivo. It can be used during a surgical or scientific procedure, and the animal may be anaesthetized or not.

Claims

Patent Claims

1. A device (1) for immobilizing an animal in order to examine a selected area of the animal's body, the device (1) comprising two upright members (2a, 2b) secured by their lower ends to a base (3), the height of the upright members (2a, 2b) being adjustable independently of one another and being adapted to be fixed at a chosen position, the device further comprising a retention bar (4) having two ends (4a, 4b), each end (4a, 4b) being releasably engageable with an upper end of one upright member (2a, 2b) by means of a connection structure (7a, 7b) enabling the retention bar (4) to be rotated around its longitudinal axis (X) and to be fixed in a chosen rotated position, an oblong support element (9) being releasably fixed to the retention bar (4) between its two ends (4a, 4b), the support element (9) having a proximal end (9a) adapted to be releasably fixed to the retention bar (4) and a distal end (9b) adapted to be releasably fixed to the animal body.

2. The device according to claim 1, characterized in that the support element (9) extends transversally to the retention bar (4) and has a longitudinal axis (Y) as well as two engagement surfaces, one at the proximal end (9a) and the other at the distal end (9b), the engagement surfaces being substantially parallel to the axis (Y) and lying in the planes that form an angle (a) ranging from 0° to 90° between each other.

3. The device according to claim 1, characterized in that each upright member (2a, 2b) is formed by a telescoping pole composed of at least two substantially concentric elements (5, 5').

4. The device according to claim 3, characterized in that the telescoping poles are fixed at a chosen vertical position by locking bolts (6a, 6b), preferably thumbscrews.

5. The device according to claim 1 or 3 or 4, characterized in that each connection structure (7a, 7b) is formed by a hollow sleeve connected transversally to the upright member (2a, 2b), each end (4a, 4b) of the retention bar (4) being located within the hollow sleeve and being held in the chosen rotated position by means of a locking bolt (8a, 8b), preferably a thumbscrew extending through the wall of the hollow sleeve.

6. The device according to claim 5, characterized in that each hollow sleeve has an internal diameter adapted to accommodate the end (4a, 4b) of the retention bar (4) with a play so that the heights of the upright members (2a, 2b) may be different.

7. The device according to claim 1 or 3 or 4, characterized in that the lower ends of the upright members (4a, 4b) are integral with the base (3).

8. The device according to claim 1, characterized in that the support element (9) is a plate-like element having a helical twist about its centre, its engagement surfaces being constituted by its regions near the ends (9a, 9b).

9. The device according to claim 1, characterized in that the support element (9) is a bar having a central part of a circular cross-section and two bilaterally recessed parts at the ends (9a, 9b) comprising the flat engagement surfaces.

10. The device according to claim 1 or 8 or 9, characterized in that the support element (9) is affixed to the retention bar (4) by means of a locking bolt (10), preferably a thumbscrew.

11. The device according to claim 2 or 8 or 9, characterized in that the angle (a) ranges from 20° to 35°.

12. The device according to claim 1, characterized in that the angle (a) is selected to match the position of the part of the animal body that it is fixed to the support element (9) and to the area of its body to be examined.

13. The device according to claim 1 or 7 or 8, characterized in that the engagement surfaces of the support element (9) are provided with apertures (12a, 12b) for receiving fixing means, preferably the apertures (12a, 12b) are threaded and the fixing means are thumbscrews.

14. The device according to claim 1, characterized in that it is sized to match the animal being worked on, particularly preferably the size of laboratory rodents.

15. The device according to claim 1, characterized in that its components are made of a material that is rigid enough to restrain the animal in position, preferably stainless steel or high-grade aluminum.

16. The device according to claim 1 or 3 or 4, characterized in that the height of each upright member (2a, 2b) is scaled in order to enable repeatable positioning of the animal.

17. The device according to claim 1, characterized in that the rotation of the retention bar (4) around its longitudinal axis (X) is scaled in order to enable repeatable positioning of the animal.

18. An assembly for immobilizing an animal in order to examine a selected area of the animal's body, the assembly comprising the device according to any of claims 1-17 and a number of exchangeable support elements (9), each support element (9) having its engagement surfaces defining a different angle (a) therebetween.

19. Use of the device according to any of claims 1 to 17 for immobilisation of an animal during a surgical or scientific procedure.

20. The use according to claim 19, characterized in that the animal is anaesthetized.

21. The use according to claim 19 or 20, characterized in that the procedure is a procedure performed under a microscope such as a stereoscope, a light microscope or a fluorescent microscope, preferably a confocal microscope, most preferably a 2P microscope, or any other microscope capable of imaging in vivo.

22. The use according to claim 19 or 20, characterized in that the procedure is performed on the animal's head, preferably on its brain, particularly preferably through a trans-cranial window (13).

23. A method of immobilising an animal by means of the device according to claims 1-17, in order to examine a selected area of the animal's its body, the animal having a fixing member (14) attached close to said selected part of its body, the method comprising:

• connecting the engagement surface at the distal end (9b) of the support element (9) to the fixing element (14) attached to the animal's its body

• connecting the engagement surface at the proximal end (9b) of the support element (9) to the retention bar (4)

• inserting the ends (4a, 4b) of the retention bar (4) in to the respective connection structures (7a, 7b)

• adjusting and fixing the height of each upright member (2a, 2b) as well as the rotation angle of the retention bar (4) to match the size and shape of the animal, so that the selected part of the animal's body is in a position enabling examination.

24. A method of immobilising an animal by means of the assembly according to claim 18, in order to examine a selected area of the animal's body, the animal having a fixing member (14) attached close to said selected part of its body, the method comprising:

• selecting a support element (9) having a suitable angle (a) and connecting it to the retention bar (4)

25. The method according to claim 24, characterized in that it further comprises exchanging the selected support element (9) with another support element (9) having a different angle (a) at least once in order to improve the positioning of the selected part of the animal's body.

26. The method according to claim 23 or 24, characterized in that the selected part of the animal's body is positioned in a way allowing access to a transcranial window for performing procedures on an animal's brain, preferably under a microscope, preferably for imaging using a 2P microscope.