CN113545337A - Method for sizing a mammalian heart valve - Google Patents
Method for sizing a mammalian heart valve Download PDFInfo
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- CN113545337A CN113545337A CN202110827373.6A CN202110827373A CN113545337A CN 113545337 A CN113545337 A CN 113545337A CN 202110827373 A CN202110827373 A CN 202110827373A CN 113545337 A CN113545337 A CN 113545337A
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- heart
- agar gel
- sizing
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/0231—Chemically defined matrices, e.g. alginate gels, for immobilising, holding or storing cells, tissue or organs for preservation purposes; Chemically altering or fixing cells, tissue or organs, e.g. by cross-linking, for preservation purposes
Abstract
The invention discloses a method for shaping a heart valve of a mammal. The method comprises the following steps: (1) injecting preheated agar gel into the cleaned mammalian heart from the aorta to fill the left ventricle and left atrium with agar gel; (2) clamping the aorta, and injecting preheated agar gel from the right ventricle into the mammalian heart in (1) to fill the right ventricle and right atrium with the agar gel; (3) immersing the mammalian heart in (2) into a pre-cooled 10% (v/v) neutral formalin buffer for cooling and fixing; the preheating temperature is 60-75 ℃; the cooling temperature is 0 ℃; the cooling time is 20-40 min; the fixed temperature is 20-25 ℃; the fixed time is 40-50 h. The invention replaces expensive Histogel by using cheap agar gelTMThe same shaping effect is obtained, the cost is greatly reduced, and the popularization and the application of the technology are facilitated.
Description
Technical Field
The invention belongs to the field of tissue organ fixing and shaping, and particularly relates to a shaping method of a mammalian heart valve.
Background
Drug non-clinical safety assessment requires detailed and systematic examination of heart valves in laboratory animals, including rats or mice. For most standard rodent trials, the heart is segmented by longitudinal cuts, including the theoretical left ventricle, right ventricle, interventricular septum, left atrium, right atrium, and the major blood vessels connecting the heart. However, due to the small heart volume, thin valve leaflets and the different material drawing/slicing methods used in rodents, the sliced samples of rodent heart valves often show inconsistent phenomena. Thus, in a slice of a heart, some slices may have one or more valves, while other slices may not have any valves. In addition, current tableting techniques often encounter difficulties in the embedding and sectioning process, such as tissue crimping, folding, and failure to obtain a complete heart valve section. In order to solve the problems, it is important to shape the heart valve before drawing materials.
As shown in fig. 1, the most common cardiac valvular sizing methods at present are: the rat heart was removed quickly after necropsy, rinsed clean and then injected with 10% Neutral formalin buffer (NBF) through the aorta using a 20g needle and 20mL syringe; clamping the aorta and immersing the intact heart in 10% NBF for three to six hours for fixation; the fixed heart was removed and injected using the following technique
Supplied Pre-heating (52 ℃ C.) HistogelTM: a quantity of tissue gel was aspirated with a 20mL syringe, a 20g gavage needle was inserted into the aorta (a of fig. 1), then a syringe needle was inserted into the right ventricle of each heart (B of fig. 1), and the gel was injected into the heart to fill the four chambers. In order to make HistogelTMThe gel was cooled to set as soon as possible, and after injection the heart was infiltrated into a pre-cooled vial of 10% NBF, placed on wet ice for 30 minutes and then removed, and placed at room temperature for 48 hours. The hearts were placed individually in acrylic, provided by Zivic instruments (Pittsburgh, Pa.)Rat heart section mould (C in fig. 1) and material selection as follows: the heart was positioned with the pulmonary artery (marker) towards the left edge of the stroma (fig. 1D). Each heart was cut at 1 mm from the pulmonary artery (E of fig. 1) and about 3 mm from the aorta (F of fig. 1), and three tissue blocks were prepared in succession. The rest of HistogelTMIs added to the cut ventricle and cooled as necessary to maintain the shape during processing. The resulting tissue block (G of fig. 1) was then placed in a separate tissue cassette, with a sponge placed between the tissue block and the cassette lid to prevent curling during handling. The cassette was placed in 70% alcohol (used for immunohistochemistry) and embedded in paraffin. See Joanna Barton et al, "Innovation in Heart Preservation to Option the Sampling and Examination of cadent Heart Valves Using the HistoGelTM Technique”，The Journal of Histotechnology,Vol.32,No.2,June 2009。
Mixing HistogelTMInjection into rodent hearts has proven to be a currently effective method to address inconsistencies in rodent heart valve drawing. With this method, less problems are encountered in tissue embedding and tissue curling and folding are reduced when sectioning. This saves both embedding time and produces a better quality tissue wax block. Thus, it is easier to obtain a section containing the entire heart valve.
By utilizing the technology, the heart can be accurately sampled, embedded and sliced, and all four valves of the rat heart can be evaluated under a microscope, so that the inconsistency among sliced samples is greatly reduced. However, HistoGelTMThe price is expensive, so that the technology cannot be popularized and popularized, and an economical and convenient shaping method needs to be developed.
Disclosure of Invention
The invention aims to overcome the defect that the prior art lacks a low-cost and effective sizing agent, and provides a sizing method of a mammalian heart valve. The setting method of the invention effectively saves the test cost and has the same effect as the prior HistogelTMCompared with the non-differential sizing effect, the method is favorable for popularization and application.
In searching for HistoGelTMIn the course of the substitute, the inventors found that the agar gel had a good fixing effect. The inventor tries to shape the heart valve of the mammal by using the agar gel, and further gropes the shaping condition, finally obtains the method for shaping the heart valve of the mammal by using the agar gel, and obtains the heart valve shaping effect and HistogelTMNo difference exists, complete histopathology evaluation can be carried out, and the cost is greatly reduced.
The invention solves the technical problems through the following technical scheme:
a first aspect of the invention provides a method of sizing a mammalian heart valve, the method comprising the steps of:
(1) injecting preheated agar gel into the cleaned mammalian heart from the aorta to fill the left ventricle and left atrium with agar gel;
(2) clamping the aorta, and injecting preheated agar gel from the right ventricle into the mammalian heart in (1) to fill the right ventricle and right atrium with the agar gel;
(3) immersing the mammalian heart in (2) into a pre-cooled 10% (v/v) neutral formalin buffer for cooling and fixing;
the preheating temperature is 60-75 ℃;
the cooling temperature is 0 ℃; the cooling time is 20-40 min;
the fixed temperature is 20-25 ℃; the fixed time is 40-50 h.
In a preferred embodiment of the present invention, the preheating temperature is 65-72 ℃.
In a specific embodiment of the invention, the temperature of the preheating is 72 ℃.
In a preferred embodiment of the present invention, the cooling time is 25-35 min.
In a specific embodiment of the invention, the cooling time is 30 min.
In a preferred embodiment of the present invention, the fixed temperature is 20-22 ℃; the fixed time is 45-50 h.
In a specific embodiment of the invention, the fixed temperature is 22 ℃; the fixed time is 48 h.
In a preferred embodiment of the invention, the agar gel may be conventional in the art, for example, a gel comprising agarose and agar pectin.
In a more preferred embodiment of the present invention, said (1) further comprises (0): the isolated mammalian heart was rinsed clean with physiological saline.
The normal saline is 0.85-0.9% sodium chloride aqueous solution; the percentage is mass to volume ratio.
In a specific embodiment of the present invention, the physiological saline is a 0.9% aqueous solution of sodium chloride; the percentage is mass to volume ratio.
In a more preferred embodiment of the present invention, said (3) is followed by (4): placing the mammalian heart in the step (3) into a heart section mould for taking materials.
The material taking method comprises the following steps:
s1: cutting the mammalian heart at a distance of 0.8-1.2 mm from the pulmonary artery;
s2: cutting the mammalian heart at 2.8-3.2 mm from the aorta.
In the present invention, the techniques used for the injection may be conventional in the art, such as injection using a syringe and needle, injection using an infusion pump.
In a second aspect, the present invention provides the use of an agar gel for the preparation of a mammalian heart valve sizing agent.
The agar gel may be conventional in the art, and is preferably a gel comprising agarose and agar pectin.
On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention.
The reagents and starting materials used in the present invention are commercially available.
The positive progress effects of the invention are as follows:
the setting method of the invention has low use priceCheap agar gel to replace expensive HistogelTMIts setting effect is similar to HistogelTMCompared with the method without difference, the four valves of the heart section obtained by taking the materials can be subjected to complete histopathology evaluation, so that the cost is greatly reduced, and the popularization and the application of the technology are facilitated.
Drawings
FIG. 1 is a schematic flow chart of the operation of a method in the background art;
in the figure: a is the injection of HistoGel into heart by inserting into aortaTM(ii) a B injecting HistoGel into heart by inserting right ventricleTM(ii) a C is an acrylic rat heart slicing die; d is the rat heart positioned in the heart section mould; e is cutting the heart at 1 mm from the pulmonary artery; f is cutting the heart about 3 mm from the aorta; g is the tissue mass taken ready for treatment.
FIG. 2 is a schematic flow chart of the operation of the present invention;
in the figure: a is perfused with normal saline to flush the heart; b, inserting into aorta and injecting agar gel into heart; c, inserting the right ventricle and injecting agar gel into the heart; d is a rat heart section mould; e is the rat heart positioned in the heart section mould; f is cutting the heart at 1 mm from the pulmonary artery; g is cutting the heart about 3 mm from the aorta; h is the tissue mass taken ready for treatment.
FIG. 3 shows the results of staining the cardiac tissue section in example 1;
in the figure: arrows in a indicate the pulmonary valve; the arrow in B indicates the aortic valve; the arrow in C indicates the mitral valve; the arrow in D indicates the tricuspid valve.
FIG. 4 shows the result of staining a cardiac tissue slice by a method of the background art;
in the figure: arrows in a indicate the pulmonary valve; the arrow in B indicates the aortic valve; the arrow in C indicates the mitral valve; the arrow in D indicates the tricuspid valve.
Detailed Description
The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.
Agar used in the examples was purchased from alatin, CAS No. 9002-18-0; rats were purchased from Zhejiang vitamin Tonglihua laboratory animals Co.
Example 1
As shown in fig. 2, the heart of the experimental rat is removed rapidly after the examination, and the heart is flushed by injecting physiological saline into the heart through a syringe and a needle of the same type (fig. 2A); sucking the pre-heated agar gel at 72 deg.C with a syringe, and injecting the gel into heart through aorta (B of FIG. 2); inserting the right ventricle and injecting the gel into the heart (fig. 2C); the heart was perfused into gel in four chambers and soaked in pre-cooled 10% neutral formalin and placed on wet ice, after cooling for fixation in 10% neutral formalin for 48 hours, the heart was placed in rat heart section molds for harvesting (fig. 2D).
In the injection of the agar gel, it was found that if a 10% neutral formalin solution is injected first and then the agar gel is injected, the chamber is not easily filled with the injected agar gel.
As shown in E of fig. 2, the material obtaining steps are as follows: cutting the heart at 1 mm from the pulmonary artery (F of fig. 2); the heart was cut about 3 mm from the aorta (G of fig. 2); placing the trimmed tissue block in an embedding box (fig. 2H), placing the sponge block between the tissue block and the embedding box cover to prevent curling during processing; the embedding box is put into 70% alcohol solution (used for immunohistochemistry), dehydrated, embedded with paraffin, and stained with hematoxylin-eosin. The results of immunostaining are shown in FIG. 3.
As shown in A-D of FIG. 3, the setting effect and Histogel of this exampleTM(FIGS. 4A-D) comparison shows no difference, and the complete histopathological evaluation was performed on all four valves of the heart section.
Claims (10)
1. A method of sizing a mammalian heart valve, comprising the steps of:
(1) injecting preheated agar gel into the cleaned mammalian heart from the aorta to fill the left ventricle and left atrium with agar gel;
(2) clamping the aorta, and injecting preheated agar gel from the right ventricle into the mammalian heart in (1) to fill the right ventricle and right atrium with the agar gel;
(3) immersing the mammalian heart in (2) into a pre-cooled 10% (v/v) neutral formalin buffer for cooling and fixing;
the preheating temperature is 60-75 ℃;
the cooling temperature is 0 ℃; the cooling time is 20-40 min;
the fixed temperature is 20-25 ℃; the fixed time is 40-50 h.
2. The sizing method according to claim 1, wherein the preheating temperature is 65-72 ℃;
preferably, the temperature of the preheating is 72 ℃.
3. The sizing method according to claim 1, wherein the cooling time is 25 to 35 min;
preferably, the cooling time is 30 min.
4. The sizing method according to claim 1, wherein the fixing temperature is 20 to 22 ℃; the fixed time is 45-50 h;
preferably, the fixed temperature is 22 ℃; the fixed time is 48 h.
5. The typing method according to claim 1, wherein the agar gel is a gel comprising agarose and agar pectin.
6. The sizing process according to claim 1, wherein (1) is preceded by (0): washing the isolated mammalian heart clean with physiological saline;
the normal saline is 0.85-0.9% sodium chloride aqueous solution; the percentage is mass to volume ratio.
7. The styling method according to claim 6 wherein the physiological saline is a 0.9% aqueous sodium chloride solution; the percentage is mass to volume ratio.
8. The sizing process according to claim 1, further comprising (4) after (3): placing the mammalian heart in the step (3) into a heart section mould for taking materials.
9. The sizing method according to claim 8, wherein the taking of the material comprises:
s1: cutting the mammalian heart at a distance of 0.8-1.2 mm from the pulmonary artery;
s2: cutting the mammalian heart at 2.8-3.2 mm from the aorta.
10. The application of agar gel in preparing a mammal heart valve sizing reagent;
preferably, the agar gel is a gel comprising agarose and agar pectin.
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