CN108175538A - A kind of Deep Hypothermia Circulatory Arrest rat animal model - Google Patents

A kind of Deep Hypothermia Circulatory Arrest rat animal model Download PDF

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Publication number
CN108175538A
CN108175538A CN201810074698.XA CN201810074698A CN108175538A CN 108175538 A CN108175538 A CN 108175538A CN 201810074698 A CN201810074698 A CN 201810074698A CN 108175538 A CN108175538 A CN 108175538A
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rat
animal model
circulatory arrest
blood
brain
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朱耀斌
李志强
刘扬
樊星
丁楠
伊寒露
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    • A61D—VETERINARY INSTRUMENTS, IMPLEMENTS, TOOLS, OR METHODS
    • A61D7/00—Devices or methods for introducing solid, liquid, or gaseous remedies or other materials into or onto the bodies of animals
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    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
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    • A61K36/53—Lamiaceae or Labiatae (Mint family), e.g. thyme, rosemary or lavender
    • A61K36/537—Salvia (sage)
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    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
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    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/71—Ranunculaceae (Buttercup family), e.g. larkspur, hepatica, hydrastis, columbine or goldenseal
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    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/73—Rosaceae (Rose family), e.g. strawberry, chokeberry, blackberry, pear or firethorn
    • A61K36/739—Sanguisorba (burnet)
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Abstract

The invention belongs to rat model technical fields, disclose a kind of Deep Hypothermia Circulatory Arrest rat animal model, prepare healthy male rat 60,350~450g of weight, measure respiratory rate, airway pressure, angiosthenia, the changes in heart rate of rat, blood is entered into venous chambers by venous drainage pipe, is pumped by the turn of tidal stream of digital controllable flow rate, blood pump is entered into miniature oxygenator；After being equipped with priming fluid and brain protection liquid, exact constant temperature bath and it is external put ice bits environment under, stop recycling 60min, brain protective tissue interruption carries out retrograde cerebral perfusion protection, then restores cycle, body surface and blood rewarming simultaneously, final measurement.The model manipulation of the present invention is simpler, spends human and material resources smaller, and the period is short, simple and reliable, economical and practical, has very big research significance.

Description

A kind of Deep Hypothermia Circulatory Arrest rat animal model
Technical field
The invention belongs to rat model technical field more particularly to a kind of Deep Hypothermia Circulatory Arrest rat animal models.
Background technology
Nineteen fifty-one, Cardiopulmonary Bypass is applied for the first time in department of cardiac surgery, with the development of Cardiopulmonary Bypass, in clinic Application it is more and more extensive.Not only in cardiothoracic surgery, but also it is applied to Giant intracranial aneurysms and blood vessel in neurosurgery The treatment of deformity.But the double action that this technology is protected in frost bite injury and brain limits the technology in the further of clinic Using.The animal model of experimental study mostly using more big animals such as pig, dog, sheep, monkeys as research object, exists both at home and abroad at present The problems such as operation is more complicated, costly, the period is long, Multidisciplinary Cooperation.
In conclusion problem of the existing technology is：The double action that this technology is protected in frost bite injury and brain, limit The technology has been made further to apply in clinical.At present both at home and abroad experimental study animal model mostly with pig, dog, sheep, monkey etc. compared with There is the problems such as operation is more complicated, costly, the period is long, Multidisciplinary Cooperation as research object in larger animal.
Invention content
In view of the problems of the existing technology, the present invention provides a kind of Deep Hypothermia Circulatory Arrest rat animal models.
The invention is realized in this way a kind of Deep Hypothermia Circulatory Arrest rat animal model is：Prepare healthy male rat 60 Only, 350~450g of weight is randomly divided into 10 groups；2 groups are compareed, 8 groups of experimental group；
Prepare a concentration of 2%, 5%, 10%, 15% brain are protected to the aqueous solution of liquid, respectively to difference two in experimental group Group is drunk 7 days~15 days；It is denoted as respectively：T1、T2、T3、T4；
In the meantime, respiratory rate, airway pressure, angiosthenia, the changes in heart rate of daily Timing measurement rat；
Blood is entered into venous chambers by venous drainage pipe, is pumped by the turn of tidal stream of digital controllable flow rate, by blood pump Enter miniature oxygenator；After being equipped with priming fluid and brain protection liquid, exact constant temperature bath and it is external put ice bits environment under, stop recycling 60min, brain protective tissue interruption carry out retrograde cerebral perfusion protection, then restore cycle, body surface and blood while rewarming, finally Measurement result.
A kind of construction method of the Deep Hypothermia Circulatory Arrest rat animal model, including：Prepare healthy rat, preparing experiment Equipment, the measurement of rat sign Index, extracorporal circulatory system, priming fluid configuration, the configuration of brain protection liquid, selective cerebral perfusion, observation result.
Further, the healthy male rat 60 of preparation healthy rat requirement, 350~450g of weight.
Further, the preparing experiment equipment has：Microfauna lung ventilator, venous chambers, cardiopulmonary bypass unit, exact constant temperature bath Slot, temperature monitor, polygraph, miniature oxygenator.
Further, the rat sign Index measurement has respiratory rate, airway pressure, angiosthenia, changes in heart rate.
Further, the extracorporal circulatory system is：Venous chambers and cardiopulmonary bypass unit, miniature oxygenator are passed through into 1.6mm silicone tubes Connection.Blood enters venous chambers by venous drainage pipe, is pumped, blood pump is entered miniature by the turn of tidal stream of digital controllable flow rate Oxygenator.
Further, the priming fluid configuration step is：After taking one anesthesia of rat, test tube of hepari takes new blood about 20ml, Gelofusine 10ml, lactated Ringer's formula liquid 10ml, heparin 1ml, sodium bicarbonate 1ml, mixed configuration.
Further, the brain protection liquid configuration step is：2.5% D-40,0.45% physiological saline 500ml, sodium bicarbonate 11mmol, calcium chloride 2,5mg, 20% mannitol 12.5mg, Li Kaduoyin 100mg, oxygen content 20ml/L, It is cooled to 4 DEG C in advance.
Further, the selective cerebral perfusion method is:Exact constant temperature bath and it is external put ice bits environment under, stop following Ring 60min, brain protective tissue interruption carry out retrograde cerebral perfusion protection, then restore cycle, body surface and blood while rewarming.
Further, the observation result is：Heartbeating recovery rate reaches 100%, and autonomous respiration recovery rate reaches 80%, routine HE is dyed and is shown no obvious abnormalities variation under transmission electron microscope observing.
Further, the rat sign Index measuring method includes：
Hybridization image is acquired with laser scanner, Array--Pro softwares are digitally converted hybridization image, data Processing and analysis calculate the p value that two groups of ratios for detecting signal and t are examined, with p<0.01 is defined as signal, and there were significant differences Property analyzes the site of differential expression.
It also needs to carry out after analyzing the site of differential expression：
Statistical procedures：According to the property of data, distribution and design feature, using SPSS13.0 statistical softwares to data into Row analysis.
Further, included with laser scanner acquisition hybridization image method：
Ultrasonography is carried out to Deep Hypothermia Circulatory Arrest rat animal model, probe used is popped one's head in for L15-7io superficials, to exhaling Frequency, airway pressure, angiosthenia, changes in heart rate Chang Huisheng records are inhaled, and image is acquired after measuring size；
Micro CT enhancing scannings are carried out to the Deep Hypothermia Circulatory Arrest rat animal model of structure, what Micro CT were used makes Shadow agent is Fenestra LC, and contrast agent dosage is 0.4ml/20g, scans the enhancement method taken every time：2 after injection contrast agent Hour is scanned.
Another object of the present invention is to provide a kind of by the pre- of Deep Hypothermia Circulatory Arrest rat animal model verification curative effect Anti- frost bite injury and the drug of brain protection, the drug is by rhizoma zingiberis 10g, garden burnet 30g, Radix Rehmanniae 15g, Chinese cassia tree 6g, dried orange peel 10g, party Join 20g, Semen Cuscutae 12g, Cortex Eucommiae 12g, teasel root 12g, rhizoma atractylodis macrocephalae 15g, Radix Glycyrrhizae 8g, ginseng 10g, cooked aconite 10g, camphor 10- 25g, cuttlebone 5-10g, vaseline 30-40g, Radix Salviae Miltiorrhizae 5-10g, Radix Astragali 2-5g, monkshood 5-10g, cassia twig 10-15g, semen brassicae 2- 5g, prepared rehmannia root 5-8g and Chinese ephedra 3-7g compositions.
Advantages of the present invention and good effect are：The model manipulation is simpler, spends human and material resources smaller, and the period is short, Simple and reliable, economical and practical, it is comprehensive to be related to comparison, and result is satisfactory, has very big research significance.
It the effect of drug that the prevention frost bite injury and brain of the model verification curative effect of the present invention are protected, is carried for developing new drug object For foundation.
The present invention is especially suitable for a variety of image technology imaging analysis such as clinical common ultrasound, CT, MRI and PET, and Easy to operate, expense is low.Diagnostic application for model provides advanced image technology.
Description of the drawings
Fig. 1 is Deep Hypothermia Circulatory Arrest rat animal model construction method flow chart provided in an embodiment of the present invention.
Specific embodiment
In order to further understand the content, features and effects of the present invention, the following examples are hereby given, and coordinate attached drawing Detailed description are as follows.
The structure of the present invention is explained in detail below in conjunction with the accompanying drawings.
Deep Hypothermia Circulatory Arrest rat animal model provided in an embodiment of the present invention is：Prepare healthy male rat 60, body 350~450g is weighed, is randomly divided into 10 groups；2 groups are compareed, 8 groups of experimental group；
Prepare a concentration of 2%, 5%, 10%, 15% brain are protected to the aqueous solution of liquid, respectively to difference two in experimental group Group is drunk 7 days~15 days；It is denoted as respectively：T1、T2、T3、T4；
In the meantime, respiratory rate, airway pressure, angiosthenia, the changes in heart rate of daily Timing measurement rat；
Blood is entered into venous chambers by venous drainage pipe, is pumped by the turn of tidal stream of digital controllable flow rate, by blood pump Enter miniature oxygenator；After being equipped with priming fluid and brain protection liquid, exact constant temperature bath and it is external put ice bits environment under, stop recycling 60min, brain protective tissue interruption carry out retrograde cerebral perfusion protection, then restore cycle, body surface and blood while rewarming, finally Measurement result.
As shown in Figure 1, the construction method of Deep Hypothermia Circulatory Arrest rat animal model provided in an embodiment of the present invention includes：
S101：Prepare healthy rat；
S102：Preparing experiment device；
S103：Rat sign Index is surveyed；
S104：It follows in vitro；
S105：Priming fluid is configured；
S106：Brain protection liquid is matched；
S107：Selective brain fills；
S108：Observe result.
The healthy male rat 60 of preparation healthy rat requirement, 350~450g of weight.
The preparing experiment equipment has：Microfauna lung ventilator, venous chambers, cardiopulmonary bypass unit, exact constant temperature bath, temperature Monitor, polygraph, miniature oxygenator.
The rat sign Index, which measures 3, respiratory rate, airway pressure, angiosthenia, changes in heart rate.
The extracorporal circulatory system is：Venous chambers are connect with cardiopulmonary bypass unit, miniature oxygenator by 1.6mm silicone tubes.Blood Liquid enters venous chambers by venous drainage pipe, is pumped by the turn of tidal stream of digital controllable flow rate, blood pump is entered miniature oxygenator.
The priming fluid configuration step is：After taking one anesthesia of rat, test tube of hepari takes new blood about 20ml, Gelofusine 10ml, lactated Ringer's formula liquid 10ml, heparin 1ml, sodium bicarbonate 1ml, mixed configuration.
Brain protection liquid configuration step is：2.5% D-40,0.45% physiological saline 500ml, bicarbonate Sodium 11mmol, calcium chloride 2,5mg, 20% mannitol 12.5mg, Li Kaduoyin 100mg, oxygen content 20ml/L are cooled to 4 DEG C in advance.
The selective cerebral perfusion method is:Exact constant temperature bath and it is external put ice bits environment under, stop recycling 60min, brain protective tissue interruption carry out retrograde cerebral perfusion protection, then restore cycle, body surface and blood while rewarming.
It is described observation result be：Heartbeating recovery rate reaches 100%, and autonomous respiration recovery rate reaches 80%, conventional H E dyeing With variation is shown no obvious abnormalities under transmission electron microscope observing.
The rat sign Index measuring method includes：
Hybridization image is acquired with laser scanner, Array--Pro softwares are digitally converted hybridization image, data Processing and analysis calculate the p value that two groups of ratios for detecting signal and t are examined, with p<0.01 is defined as signal, and there were significant differences Property analyzes the site of differential expression.
It also needs to carry out after analyzing the site of differential expression：
Statistical procedures：According to the property of data, distribution and design feature, using SPSS13.0 statistical softwares to data into Row analysis.
Further, included with laser scanner acquisition hybridization image method：
Ultrasonography is carried out to Deep Hypothermia Circulatory Arrest rat animal model, probe used is popped one's head in for L15-7io superficials, to exhaling Frequency, airway pressure, angiosthenia, changes in heart rate Chang Huisheng records are inhaled, and image is acquired after measuring size；
Micro CT enhancing scannings are carried out to the Deep Hypothermia Circulatory Arrest rat animal model of structure, what Micro CT were used makes Shadow agent is Fenestra LC, and contrast agent dosage is 0.4ml/20g, scans the enhancement method taken every time：2 after injection contrast agent Hour is scanned.
The embodiment of the present invention provides a kind of prevention frostbite by Deep Hypothermia Circulatory Arrest rat animal model verification curative effect Damage and brain protection drug, the drug by rhizoma zingiberis 10g, garden burnet 30g, Radix Rehmanniae 15g, Chinese cassia tree 6g, dried orange peel 10g, Radix Codonopsis 20g, Semen Cuscutae 12g, Cortex Eucommiae 12g, teasel root 12g, rhizoma atractylodis macrocephalae 15g, Radix Glycyrrhizae 8g, ginseng 10g, cooked aconite 10g, camphor 10-25g, sea Piao Octopus 5-10g, vaseline 30-40g, Radix Salviae Miltiorrhizae 5-10g, Radix Astragali 2-5g, monkshood 5-10g, cassia twig 10-15g, semen brassicae 2-5g, cultivated land Yellow 5-8g and Chinese ephedra 3-7g compositions.
The present invention prepares healthy male rat 60,350~450g of weight, measure the respiratory rate of rat, airway pressure, Blood is entered venous chambers by angiosthenia, changes in heart rate by venous drainage pipe, is pumped by the turn of tidal stream of digital controllable flow rate, Blood pump is entered into miniature oxygenator.Priming fluid is equipped with after brain protection liquid, environment is considered to be worth doing in exact constant temperature bath and external ice of putting Under, stop recycling 60min, brain protective tissue interruption carries out retrograde cerebral perfusion protection, then restores cycle, body surface and blood are simultaneously Rewarming, final measurement.
The model manipulation is simpler, spends human and material resources smaller, and the period is short, simple and reliable, economical and practical, is related to comparing Comprehensively, and result is satisfactory, has very big research significance.
The above is only the preferred embodiments of the present invention, and not makees limitation in any form to the present invention, Any simple modification made to the above embodiment of every technical spirit according to the present invention, equivalent variations and modification, belong to In the range of technical solution of the present invention.

Claims (7)

1. a kind of Deep Hypothermia Circulatory Arrest rat animal model, which is characterized in that the Deep Hypothermia Circulatory Arrest rat animal model is： Prepare healthy male rat 60,350~450g of weight is randomly divided into 10 groups；2 groups are compareed, 8 groups of experimental group；
Prepare a concentration of 2%, 5%, 10%, 15% brain are protected to the aqueous solution of liquid, drunk respectively to different two groups in experimental group With 7 days~15 days；It is denoted as respectively：T1、T2、T3、T4；
In the meantime, respiratory rate, airway pressure, angiosthenia, the changes in heart rate of daily Timing measurement rat；
Blood is entered into venous chambers by venous drainage pipe, is pumped by the turn of tidal stream of digital controllable flow rate, by blood pump in a subtle way Type oxygenator；After being equipped with priming fluid and brain protection liquid, exact constant temperature bath and it is external put ice bits environment under, stop recycling 60min, brain protective tissue interruption carry out retrograde cerebral perfusion protection, then restore cycle, body surface and blood while rewarming, finally Measurement result.
A kind of 2. construction method of Deep Hypothermia Circulatory Arrest rat animal model as described in claim 1, which is characterized in that the depth The construction method of hypothermia circulatory arrest rat animal model includes：Prepare healthy rat, preparing experiment equipment, rat sign Index to survey Amount, extracorporal circulatory system, priming fluid configuration, the configuration of brain protection liquid, selective cerebral perfusion, observation result.
3. the construction method of Deep Hypothermia Circulatory Arrest rat animal model as claimed in claim 2, which is characterized in that described to prepare to be good for The healthy male rat 60 of health rat requirement, 350~450g of weight；
The preparing experiment equipment has：Microfauna lung ventilator, venous chambers, cardiopulmonary bypass unit, exact constant temperature bath, temperature monitoring Instrument, polygraph, miniature oxygenator；
The rat sign Index measurement has respiratory rate, airway pressure, angiosthenia, changes in heart rate；
The extracorporal circulatory system is：Venous chambers are connect with cardiopulmonary bypass unit, miniature oxygenator by 1.6mm silicone tubes.Blood leads to It crosses venous drainage pipe and enters venous chambers, pumped by the turn of tidal stream of digital controllable flow rate, blood pump is entered into miniature oxygenator；
The priming fluid configuration step is：After taking one anesthesia of rat, test tube of hepari takes new blood about 20ml, Gelofusine 10ml, Lactated Ringer's formula liquid 10ml, heparin 1ml, sodium bicarbonate 1ml, mixed configuration；
Brain protection liquid configuration step is：2.5% D-40,0.45% physiological saline 500ml, sodium bicarbonate 11mmol, calcium chloride 2,5mg, 20% mannitol 12.5mg, Li Kaduoyin 100mg, oxygen content 20ml/L are cooled to 4 DEG C in advance；
The selective cerebral perfusion method is：Exact constant temperature bath and it is external put ice bits environment under, stop recycling 60min, brain Protective tissue interruption carries out retrograde cerebral perfusion protection, then restores cycle, body surface and blood while rewarming；
It is described observation result be：Heartbeating recovery rate reaches 100%, and autonomous respiration recovery rate reaches 80%, conventional H E dyeing and thoroughly Variation is shown no obvious abnormalities under radio sem observation.
4. the construction method of Deep Hypothermia Circulatory Arrest rat animal model as claimed in claim 2, which is characterized in that the rat body Sign index measurement method includes：
Hybridization image is acquired with laser scanner, Array--Pro softwares are digitally converted hybridization image, data processing And analysis, the p value that two groups of ratios for detecting signal and t are examined is calculated, with p<There were significant differences property that 0.01 is defined as signal, point Analyse the site of differential expression.
5. the construction method of Deep Hypothermia Circulatory Arrest rat animal model as claimed in claim 4, which is characterized in that analysis difference table It also needs to carry out behind the site reached：
Statistical procedures：According to the property of data, distribution and design feature, data are divided using SPSS13.0 statistical softwares Analysis.
6. the construction method of Deep Hypothermia Circulatory Arrest rat animal model as claimed in claim 4, which is characterized in that use laser scanning Instrument acquisition hybridization image method includes：
Ultrasonography is carried out to Deep Hypothermia Circulatory Arrest rat animal model, probe used is popped one's head in for L15-7io superficials, to breathing frequency Rate, airway pressure, angiosthenia, changes in heart rate Chang Huisheng records, and acquire image after measuring size；
Micro CT enhancing scannings, the contrast agent that Micro CT are used are carried out to the Deep Hypothermia Circulatory Arrest rat animal model of structure For Fenestra LC, contrast agent dosage is 0.4ml/20g, scans the enhancement method taken every time：2 hours after injection contrast agent It is scanned.
7. the prevention frost bite injury and brain of a kind of verification of the Deep Hypothermia Circulatory Arrest rat animal model as described in claim 1 curative effect are protected The drug of shield, which is characterized in that the drug by rhizoma zingiberis 10g, garden burnet 30g, Radix Rehmanniae 15g, Chinese cassia tree 6g, dried orange peel 10g, Radix Codonopsis 20g, Semen Cuscutae 12g, Cortex Eucommiae 12g, teasel root 12g, rhizoma atractylodis macrocephalae 15g, Radix Glycyrrhizae 8g, ginseng 10g, cooked aconite 10g, camphor 10-25g, sea Piao Octopus 5-10g, vaseline 30-40g, Radix Salviae Miltiorrhizae 5-10g, Radix Astragali 2-5g, monkshood 5-10g, cassia twig 10-15g, semen brassicae 2-5g, cultivated land Yellow 5-8g and Chinese ephedra 3-7g compositions.
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