CN104568542A - Method for fixing fish brain tissue specimen through systemic heart perfusion - Google Patents
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Abstract
The invention discloses a method for fixing a fish brain tissue specimen through systemic heart perfusion. The method is mainly characterized in that after fish anesthesia, thoracotomy is performed on the periphery of a heart surface projection area of the fish, ventral aorta intubation is performed through the ventricle, common cardinal vein intubation is performed through the atrium, ligation with an operation thread is used for fixing, and an out flow tube is connected with a graduated vessel for measuring liquid out-flowing quantity; a constant-speed perfusion pump is used for perfusing normal saline through a perfusion tube, and a 10% formalin solution is adopted for reperfusion; when perfusion is started, a line is marked on the skull instantly and craniotomy is performed, and the perfusion time is then decided through combination of the perfusion quantity and the out-flowing quantity when the color of the brain tissue changes white; the brain is picked and placed in the 10% formalin solution to be fixed for 48 h after perfusion is stopped, and the brain tissue specimen with complete structure, normal form and good state is obtained. By means of the method, the problems of insufficient fixing, non-comprehensive fixing area, tissue autolysis, cell deformation and the like of the fish brain tissue can be solved effectively, the brain tissue specimen with more complete structure and more ideal state is obtained easily, and the natural form is retained better.
Description
Technical field
The invention belongs to biological field, particularly a kind of animal tissue specimens fixing means.
Background technology
Cardiac perfusion method is the technical foundation of animal brain being carried out to the researchs such as tissue slice, Pathologic specimen making, SABC, in situ hybridization, In situPCR, brain inner position labelling.Animal heart in vivo Perfusion is fixing means before a kind of tissue specimen, is in the dopey situation of animal, carries out the method for cardiac perfusion with fixative.
Animal brain is after death because autolyze phenomenon is understood in the effect of lysosomal enzyme in cell very soon, the object of cardiac perfusion is that before animal dead, apply fixative is fixed cerebral tissue, suppress the breeding of antibacterial in tissue, prevent corruption, avoid the generation of cerebral tissue autolysis, can also increase and organize hardness, make to organize not yielding, maintain original histology and morphology structure, obtain the cerebral tissue that form is complete and in good condition, and the tissue after fixing has different affinitys to dyestuff, different refractive indexs can be produced after dyeing, color is more Clear & Transparent, be convenient to observe sclerotic tissue, more be conducive to the observation of tissue slice.
Current heart in vivo Perfusion is applied to terraria mostly.
(the Ji Zhengjian such as Ji Zhengjian, Zhang Dongxia, Zhang Libo, Deng. rat is in the comparison [J] of body cerebral tissue perfusion. laboratory animal and comparative medicine, 2009,29 (6): 390-392.) in different ways rat is carried out to the comparative study of perfusion, filter out best Perfusion, i.e. apex of the heart inserting needle ligation ventral aorta group, inserts people's perfusion pin, by left ventricle until aorta from apex of the heart position towards aorta; Open abdominal cavity, ligation ventral aorta; Cut off right auricle, insert polyethylene tube, in order to liquid return.
Patent No. CN 1336540A discloses a kind of method adopting heart perfusion to remove the cell suspension of erythrocyte preparation dispersion in tissue, and adopt from left ventricle apex of the heart injection perfusate mice, the method flowed out from right ventricle carries out perfusion.
(the Liu Naihui such as Liu Naihui, Zhang Li, Shao Ying, Deng. the application of heart in vivo Perfusion in rat cerebral tissue's sections observation [J]. Laboratory Animal Science and management, 2005 (2), 52-53.) select SD rat to carry out intraperitoneal injection of anesthesia, open abdominal cavity, insert perfusion pin from left ventricle and fix, cutting off right auricle, pour into constant flow pump.
Hu Ping etc. (Hu Ping; outstanding family Luk, Luo Zhengyao. THE MODEL OF RAT HEART REPERFUSION SYNDROME [J]. Hunan medical college journal, 1986,01:15-18.) remove ligation by the main Zhi Ranhou of ligation rat left coronary artery, replicate rat heart in vivo reperfusion syndrome model.
(the Li Jing such as Li Jing, Xiao Jiasi, Hou Min. burn on the impact [J] of guinea pig in vitro heart perfusion systolic and diastolic function. Third Military Medical University's journal, 1990,06:480-483.) utilize the hollow isolated perfused heart model views burn of direct motion on the impact of heart contraction and diastolic function.
Ma Xiaole (Ma Xiaole. the research [D] that Gansu zokor brain map builds. Shaanxi Normal University, 2009.) adopt from left ventricle insertion aorta in the research of structure zokor brain map, the mode of right atrium opening has carried out cardiac perfusion, and the amount injecting fixative is about 1.5 times of body weight.
Known by more above-mentioned document cardiac perfusion method, because the species difference of animal is different with research purpose, cause the method for cardiac perfusion also different.
Aquatic animal and terraria have larger form and differences of Physiological, for the rare reported in literature of cardiac perfusion technology of Fish.For Cyprinus carpio, Cyprinus carpio heart only has a ventricle and an atrium, blood circulation is single cycle, without body circulation with pulmonary circulation point, its blood circulation route is different from other animals, also the difference to some extent such as the fixative of inserting needle position and method and use, and Cyprinus carpio is also different from the anesthesia of the terraria such as rat, gecko, and therefore cardiac perfusion techniqueflow must be different.
Summary of the invention
The object of the present invention is to provide and a kind ofly can solve employing heart in vivo perfusion that fish brain specimen fixes tissue automatic soup-dissolving's problem of middle existence to the fixing method of Fish cerebral tissue specimen.
Technical scheme of the present invention is mainly: fish is anaesthetized by dipping method, open chest surgery, perfusion pin inserted abdominal aorta through ventricle and carries out intubate and ligation is fixed; Drainage needle is carried out common cardinal vein intubate through atrium and ligation is fixed; The effuser that drainage needle connects is put into meter glass, and real time measure also monitors the volume of infusion liquid and effluent; When cardiac perfusion starts, skull draws four linear rectangularities, does rectangular cutout, open cranial cavity, expose brain; When fish brain color bleaches and volume increases to some extent, and decide infusion time in conjunction with the amount of infusion liquid and effluent; After stopping perfusion, win fish brain and be placed in fixative and be fixed.
Concrete steps of the present invention are as follows:
(1) anesthetized animal: adopt dipping method anesthesia fish, steep in the water of fish activity by anaesthetic, anaesthetic will be absorbed by the gill portion of fish and enter into blood, realizes calmness or the anesthesia of fish.
(2) open chest surgery: in conjunction with physiology and the anatomical features of fish, breast is opened at the heart body surface projection areas adjacent of fish, forward through pectoral fin cut to lower jaw place along ventrimeson with scalpel from fish belly fin front end and body axle vertical direction intersection point, spinal column is cut along the opening side of supporting or opposing before abdomeinal fin again with shears, cut after the gill cover along spinal column lower edge, removing left muscles, opens the heart body surface projection region in thoracic cavity, retain the gill cover, expose ventricle, abdominal aorta, atrium, common cardinal vein.
(3) two-tube intubate: carry out transventricular abdominal aorta intubate, fixes perfusion pin with surgical thread ligation abdominal aorta, carries out common cardinal vein intubate through atrium, and surgical thread ligation common cardinal vein fixes drainage needle.
(4) cardiac perfusion: complete intubate and fixing after, again fish is fixed, the perfusion tube that perfusion pin connects is led to constant speed charge pump, the effuser that drainage needle connects is put into graduated vessels, real time measure also monitors the volume of infusion liquid and effluent, open constant speed charge pump and carry out cardiac perfusion, first pour into normal saline displacement blood, rear perfusion concentration is 10% formalin solution.
(5) operation of opening cranium: after cardiac perfusion starts, according to construction features and the size of fish skull, immediately with fish mouth to head and trunk junction and head and trunk first fish scale point of interface for reference point, with the standardized straight line of head median line vertical direction blunt, with left and right eye socket top edge for benchmark, be parallel to each standardized straight line of median line, with left and right eye socket back edge for reference point is perpendicular to the standardized line of median line, article four, straight line intersection forms rectangle, on skull, rectangular cutout is made along four straight lines, open cranial cavity, remove cerebrospinal fluid and the fatty tissue on brain surface, expose cerebral tissue.
(6) perfusion is stopped: when the color observing fish brain bleaches and volume increases to some extent, when the amount of the formalin of perfusion is 1.5 times of fish body weight, stop perfusion, win fish cerebral tissue immediately and be positioned in 10% formalin solution and carry out specimen and fix 48 hours.
The present invention compared with prior art tool has the following advantages:
1, can effectively solve fixing insufficient, the fixed area of Fish cerebral tissue not comprehensively, tissue automatic soup-dissolving, the problem such as cytomorphosis, more complete and the state more preferably cerebral tissue specimen of easy acquisition structure, its natural form can be retained better, the aspect such as making, dyeing, labelling of brain tissue slice can be applied to very well;
2, can effectively avoid Massive Bleeding, damage excessive, can comparatively be easy to search out catheter position;
Perfusion pin and drainage needle can effectively be fixed by the intubate 3, adopted and ligating methods, prevent from occurring coming off when mobile fish body;
4, carry out out in perfusing course cranium observe can judge perfusion effect more accurately and can after perfusion stops rapid extraction brain specimen, can effectively determine perfusion dwell time in conjunction with the color of trickle and discharge.
5, the application that can be the aspects such as tissue slice, Pathologic specimen making, SABC, in situ hybridization, In situPCR, brain inner position labelling provides new technique.
Accompanying drawing explanation
Fig. 1 is Cyprinus carpio cardiac perfusion lab diagram;
Fig. 2 is the brain tissue slice figure made of the non-cardiac perfusion of conventional method;
Fig. 3 is the brain tissue slice figure carrying out cardiac perfusion making with the present invention;
Detailed description of the invention
Embodiment 1
Laboratory animal: healthy adult Cyprinus carpio, body weight 1.13kg, the long 36.52cm of body, is purchased from fish market, Qinhuangdao.
Main agents: eugenol (Dentistry Material Plant, Shanghai Medical Machinery Co., Ltd., lot number 201103), normal saline, distilled water, dimethyl diaminophenazine chloride, neutral gum, paraffin, dimethylbenzene, dehydrated alcohol, 10% formalin solution, 2% hydrochloric acid solution.
Key instrument: BQ50S micrometeor speed-regulating type peristaltic pump (Baoding Lei Fu fluid Science and Technology Ltd.), 7002 mould electric grinds (Yongkang City Xi Kelai Tools Co., ltd), RM2235 type histotome (German Leica company), TS100 type inverted biological microscope (Japanese Nikon company), HH-S type digital display thermostat water bath (Medical Instruments factory of Jintan City), tube for transfusion 2 overlap, operating theater instruments 1 is overlapped.
1, Animal Anesthesia: eugenol 6mL is mixed with dehydrated alcohol 48mL and shakes up, leave standstill 10min, pour in the box that 18L water is housed, being mixed with concentration is 0.36g/L eugenol anesthetic solution, Cyprinus carpio is placed in anesthetic solution and carries out dipping, anesthesia duration is about about 10min, enters anesthesia A3 phase (anaesthetizing for three phases) state.
2, open chest surgery: open breast around the heart body surface projection district of Cyprinus carpio, lower jaw is switched to forward at two abdomeinal fin front end edge ventrimeson with scalpel, cut to spinal column with the shears side of supporting or opposing again, cut after the gill cover along spinal column lower edge, retain the gill cover, remove left muscles, open the heart body surface projection region in thoracic cavity, expose ventricle 3., abdominal aorta, atrium 2., common cardinal vein.
3, two-tube intubate: as shown in Figure 1,3. perfusion pin is crossed bulbus arteriosus through ventricle and 1. inserts abdominal aorta and carry out intubate 5., perfusion pin is fixed in abdominal aorta by surgical thread ligation; 4. 2. drainage needle is carried out common cardinal vein intubate through atrium, and drainage needle is fixed in common cardinal vein by surgical thread ligation.
4, cardiac perfusion: the perfusion tube that perfusion pin connects is led to BQ50S micrometeor speed-regulating type peristaltic pump, puts into meter glass by the effuser that drainage needle connects, and real time measure also monitors the volume of infusion liquid and effluent.Fish is fixed, opens the constant speed perfusion that BQ50S micrometeor speed-regulating type peristaltic pump carries out heart, first with normal saline displacement blood, more slowly pour into 10% formalin solution.
5, operation of opening cranium: when cardiac perfusion starts, according to construction features and the size of Cyprinus carpio skull, immediately with fish mouth to head and trunk junction and head and trunk first fish scale point of interface for reference point, with the standardized straight line of head median line vertical direction blunt, with left and right eye socket top edge for benchmark, be parallel to each standardized straight line of median line, with left and right eye socket back edge for reference point is perpendicular to the standardized line of median line, article four, straight line intersection region forms rectangle, on skull, rectangular cutout is made according to these four straight lines, open cranial cavity, remove cerebrospinal fluid and the fatty tissue on brain surface, expose cerebral tissue.
6, stop perfusion: when the color observing fish brain bleaches and volume increases to some extent, when the amount of the paraformaldehyde solution poured into is 1.5 times of fish body weight, stop perfusion.
7, specimen is fixed: after stopping perfusion, win fish brain immediately and be placed in 10% formalin solution, fixing 48h.
8, microsection manufacture: application conventional method carries out making and the H.E dyeing of cerebral tissue paraffin section, application TS100 type inverted biological microscope is observed.
Compared with obtaining brain specimen with conventional method, the present invention carries out the brain specimen structural integrity of cardiac perfusion making, and form is normal, and fully fixing, quality is more tough.Compared with the brain tissue slice (Fig. 2) obtained with conventional method, it is clear that the present invention carries out cell in the brain tissue slice (Fig. 3) of cardiac perfusion making, and normally, acellular necrosis and tissue automatic soup-dissolving, section color is more distinct for form.
Embodiment 2
Laboratory animal: healthy adult Carassius auratus, body weight 0.33kg, the long 19.21cm of body, is purchased from fish market, Qinhuangdao.
Main agents: eugenol (Dentistry Material Plant, Shanghai Medical Machinery Co., Ltd., lot number 201103), normal saline, distilled water, dehydrated alcohol, 10% formalin solution.
Key instrument: BQ50S micrometeor speed-regulating type peristaltic pump (Baoding Lei Fu fluid Science and Technology Ltd.), 7002 mould electric grinds (Yongkang City Xi Kelai Tools Co., ltd), tube for transfusion 2 overlap, operating theater instruments 1 is overlapped.
1, Animal Anesthesia: use 0.36g/L eugenol anesthetic solution to anaesthetize, anesthesia duration about 5min (build is less, and anesthesia duration is shorter than Cyprinus carpio), enter anesthesia A3 phase (anaesthetizing for three phases) state.
2, open chest surgery: operation technique is identical with embodiment 1, and due to Carassius auratus build less, need fish body to fixedly secure in operative process to reduce the operation inconvenience caused owing to sliding.
3, two-tube intubate: intubation procedure is identical with embodiment 1, Carassius auratus build is less, its abdominal aorta and common cardinal vein length also shorter, when carrying out intubate, syringe needle intubating length is shortened in order to avoid insert broken by blood vessel.
4, cardiac perfusion: perfusing course is identical with embodiment 1, because the build of Carassius auratus is less, its body fluid volume is less equally, and slow down during perfusion perfusion rate, enough to ensure the operation of opening cranium time.
5, operation of opening cranium, stopping perfusion, specimen fixation procedure are all with embodiment 1.
In embodiment 1, Cyprinus carpio build used is comparatively large, and in the present embodiment, Carassius auratus build used is less, health is narrower, and specific implementation process is more difficult compared with embodiment 1, but apply heart in vivo perfusion of the present invention, the brain specimen structural integrity obtained, form is normal, fully fixing.
Claims (7)
1. the method adopting heart in vivo perfusion to fix Fish cerebral tissue specimen, is characterized in that: it comprises the following steps: the first step, anesthetized animal; Second step, open chest surgery; 3rd step, two-tube intubate; 4th step, cardiac perfusion; 5th step, operation of opening cranium; 6th step, stops perfusion.
2. employing heart in vivo perfusion according to claim 1 is to the fixing method of Fish cerebral tissue specimen, it is characterized in that: described anesthetized animal adopts dipping method anesthesia fish, steep in the water of fish activity by anaesthetic, anaesthetic will be absorbed by the gill portion of fish and enter into blood, realizes calmness or the anesthesia of fish.
3. employing heart in vivo perfusion according to claim 1 is to the fixing method of Fish cerebral tissue specimen, it is characterized in that: described open chest surgery is physiology in conjunction with fish and anatomical features, breast is opened at the heart body surface projection areas adjacent of fish, forward through pectoral fin cut to lower jaw place along ventrimeson with scalpel from fish belly fin front end and body axle vertical direction intersection point, spinal column is cut along the opening side of supporting or opposing before abdomeinal fin again with shears, cut after the gill cover along spinal column lower edge, removing left muscles, open the heart body surface projection region in thoracic cavity, retain the gill cover, expose ventricle, abdominal aorta, atrium, common cardinal vein.
4. employing heart in vivo perfusion according to claim 1 is to the fixing method of Fish cerebral tissue specimen, it is characterized in that: described two-tube intubate carries out transventricular abdominal aorta intubate, perfusion pin is fixed with surgical thread ligation abdominal aorta, carry out common cardinal vein intubate through atrium, surgical thread ligation common cardinal vein fixes drainage needle.
5. employing heart in vivo perfusion according to claim 1 is to the fixing method of Fish cerebral tissue specimen, it is characterized in that: described cardiac perfusion be complete intubate and fixing after, again fish is fixed, the perfusion tube that perfusion pin connects is led to constant speed charge pump, the effuser that drainage needle connects is put into graduated vessels, real time measure also monitors the volume of infusion liquid and effluent, open constant speed charge pump and carry out cardiac perfusion, first pour into normal saline displacement blood, rear perfusion concentration is 10% formalin solution.
6. employing heart in vivo perfusion according to claim 1 is to the fixing method of Fish cerebral tissue specimen, it is characterized in that: described operation of opening cranium is after cardiac perfusion starts, according to construction features and the size of fish skull, immediately with fish mouth to head and trunk junction and head and trunk first fish scale point of interface for reference point, with the standardized straight line of head median line vertical direction blunt, with left and right eye socket top edge for benchmark, be parallel to each standardized straight line of median line, with left and right eye socket back edge for reference point is perpendicular to the standardized line of median line, article four, straight line intersection forms rectangle, on skull, rectangular cutout is made along four straight lines, open cranial cavity, remove cerebrospinal fluid and the fatty tissue on brain surface, expose cerebral tissue.
7. employing heart in vivo perfusion according to claim 1 is to the fixing method of Fish cerebral tissue specimen, it is characterized in that: described stopping perfusion is when the color observing fish brain bleaches and volume increases to some extent, when the amount of the formalin of perfusion is 1.5 times of fish body weight, stop perfusion, win fish cerebral tissue immediately and be positioned in 10% formalin solution and carry out specimen and fix 48 hours.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104880546A (en) * | 2015-04-28 | 2015-09-02 | 中国科学院近代物理研究所 | Sample preparation method for mice brain tissue proteome analysis |
| CN107647931A (en) * | 2017-11-09 | 2018-02-02 | 燕山大学 | A kind of fish fixing device for being used to before brain tissue sample fix and implementation |
| CN108606854A (en) * | 2018-04-20 | 2018-10-02 | 中国水产科学研究院东海水产研究所 | A method of blood in the removal fish gill |
| CN110327132A (en) * | 2019-08-19 | 2019-10-15 | 四川农业大学 | A kind of easy sturgeon telocoele medication |
| CN111700062A (en) * | 2020-06-19 | 2020-09-25 | 山东中医药大学 | Intelligent rodent heart perfusion device |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104880546A (en) * | 2015-04-28 | 2015-09-02 | 中国科学院近代物理研究所 | Sample preparation method for mice brain tissue proteome analysis |
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| CN107647931A (en) * | 2017-11-09 | 2018-02-02 | 燕山大学 | A kind of fish fixing device for being used to before brain tissue sample fix and implementation |
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| CN110327132A (en) * | 2019-08-19 | 2019-10-15 | 四川农业大学 | A kind of easy sturgeon telocoele medication |
| CN111700062A (en) * | 2020-06-19 | 2020-09-25 | 山东中医药大学 | Intelligent rodent heart perfusion device |
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Application publication date: 20150429 |