CN102784012B - Blood brain barrier pharmacokinefic continuous dosing system and detecting system - Google Patents
Blood brain barrier pharmacokinefic continuous dosing system and detecting system Download PDFInfo
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- CN102784012B CN102784012B CN201210072127.5A CN201210072127A CN102784012B CN 102784012 B CN102784012 B CN 102784012B CN 201210072127 A CN201210072127 A CN 201210072127A CN 102784012 B CN102784012 B CN 102784012B
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Abstract
The invention discloses a detecting system capable of detecting whether drugs permeate through blood brain barrier in real time, and particularly discloses a continuous dosing device and a pharmacokinefic system which continuously extracts cerebrospinal fluid to perform high performance liquid chromatography (HPLC) analysis to detect blood brain barrier in real time under the waking state of an experimental animal. The detecting system overcomes shortcomings of existing monitoring means, is simple in operation and high in sensitivity, and has wide medical application prospects.
Description
Technical field
The invention belongs to medical detecting system field, relate to the detection system of a set of real-time detection of drugs through blood brain barrier, be specifically related to a kind of blood brain barrier pharmacokinetics to continue medication system and detection system, particularly relate to one and be enclosed within the sustained delivery device under rat waking state and continue to extract cerebrospinal fluid and carry out HPLC and analyze with the Pharmacokinetic System detecting blood brain barrier in real time.
Background technology
Brain barrier is the structure maintaining neuron surrounding microenvironment stability in central nervous system, thus ensures that neuron normal function is movable.According to the difference of form, brain barrier divides three classes: blood-brain barrier (BBB), blood-cerebrospinal fluid barrier, cerebrospinal fluid-brain barrier.Wherein, blood brain barrier is most important ingredient in three.Blood brain barrier plays a part the strict exterior materials processed of limit and enters cerebral tissue nervous system and to maintain central nervous system's microenvironment stable.Can medicine be of great significance the distribution in vivo of drugs, medication amount effect relationship tool through blood brain barrier, it is also whether medicine acts on a neuronic important indicator in central nervous system through degree, therefore, can evaluate medicine be become an important topic in materia medica research through blood brain barrier and through degree, is also the difficult problem that current medical scholar faces simultaneously.
At present, can detection of drugs mainly contain four large classes by the method for blood brain barrier: living animal method, in vitro cerebral tissue method, set up in vitro blood-brain barrier model, founding mathematical models.Can the suitable detection method of the different choice of experimentally animal in specific experiment.
Living animal detection method is evaluated medicine and is mainly divided into four classes by the method for blood brain barrier situation: the first, monitor cerebral spinal fluid (CSF).Cerebrospinal fluid-brain barrier can make CSF and the mutual traffic of extracellular fluid, now think that material is once enter CSF from blood, just cerebral tissue can be entered by free diffusing, monitoring CSF is the important means of examination by the medicine of blood brain barrier, therefore reflects neuron living environment in brain with the change of CSF for many years always.The second, brain microdialysis method is a kind of continous pouring medicine under animal waking state and gathers the method for specific brain regions district perfusate.Because this technology makes a collection of specimens through semipermeable membrane, specimen is substantially free of the materials such as macro-molecular protein, and therefore specimen directly can carry out HPLC analysis.The deficiency of this detection method is its extract is only through the dialysis of probe semipermeable membrane and the small-molecule substance of next local cells interstitial fluid, cannot reflect whole IC situation; 3rd, medicine radioactive label method is whether a kind of irradiation image method detection of drugs that utilizes can the method for penetrating blood brain barrier.As the magnetic nano-particle through rat tail vein injection 99Tc labelling, and rat head is placed in magnetic field, then observed by single photon emission computed tomography instrument, testing result can show that this carrier system successfully can carry 99Tc and stride across normal rat blood brain barrier arrival brain; 4th, carrying out medicine at living animal also can the ability of indirect proof agent permeates therethrough blood brain barrier for parametric measurement.The observation monitoring of living animal can make full use of animal economy regulatory mechanism, is equal to physiological situation when medicine is actual to be used.But the method is owing to being limited to time, funds, technical conditions etc., and be difficult to carry out large-scale medicine examination, it crosses the physiology of blood brain barrier to medicine, the observational study of pathomechanism exists limitation.
Five kinds: the first is had, single carotid injection technology by the method for in vitro cerebral tissue checking and appraising agent permeates therethrough blood brain barrier situation.After animal carotid artery puts pipe, the Ringer's solution bolus being dissolved with medicine to be checked and having carried out radiolabeled internal reference thing is entered common carotid artery, broken end, gets brain, carries out analytical calculation.This technical operation is fast, is suitable for high flux examination, but because of unexecuted outer arteries ligation in experiment, target compound can be diffused into whole body, and due to time restriction, technical difficulty is larger; The second, In situ brain perfusion.Operation needs ligation External Carotid Artery Branch occipital artery, superior thyroid artery and ligation internal carotid artery pterygoid process arteria palatina.Through external carotid artery loading, to press from both sides during input and close common carotid artery, continue input normal saline after loading with lavation.This technique sensitive is high, accurately can study the factors such as pH value, ion, flow velocity, albumen quality concentration enter brain impact on material absorbing.But the method technical operation difficulty is large, complete the required size of animal of experiment many, analysis of experimental data workload is large.3rd, intravenous injection technology.This technology is described as " goldstandard " of evaluating BBB permeability.Rat or mice inject target compound and Plasma volumes label through femoral vein or tail Vein Tube, get blood through femoral artery put pipe in different time points.Experiment is finished, and kills animal, surveys compound concentration in brain.This operates the detection technique be considered to closest to medicine real conditions in human body.4th, autoradiographic technique.This technology imports in organism by radioisotope labeled compound, through after a period of time, specimen is made section or smear, the radioactive exposure process of certain hour, then through the black Argent grain of development, the reduction of fixing processes and displays, the key sample acceptance of the bid note accurate location of thing and concentration.But have this method to there is a defect, actually or namely cannot differentiate the development of its metabolite of target compound.5th, efficient liquid phase chromatographic analysis, it is high that this technology has separation efficiency, and selectivity is good, and analysis speed is fast, sensitivity high, detects in analysis be widely used at material.
Application in vitro blood-brain barrier model (blood-brain barrier model, BBBM) checking and appraising medicine is by the method for BBB: recently cerebrovascular broken section of BBBM is widely used in an experiment, also can preserve for a long time and retains have a large amount of live body BBB feature (as adhesion complex, imitated vesicle structure, compact siro spinning technology transport son, abc transport albumen, Leptin receptor etc.) by the in vitro BBBM cultivating out.BBBM can replace living animal in an experiment because of it, time saving and energy saving, and avoids the dispute in ethics, therefore at drugs by being used widely in the kinetics of BBB.But it also has limitation, transporter as special in BBB in incubation is expressed and is weakened gradually, lacks the nerve-body fluid regulatory mechanism etc. of animal economy.
Evaluating medicine is very difficult by blood brain barrier and by degree, existing detection means has its Pros and Cons, and different medicines also often presents different drug distribution, therefore, which kind of method is not had to go for all drug distribution evaluations in existing detection means, thus research and develop high, easy and simple to handle being become by the technical approach of blood brain barrier of a kind of widely applicable, efficiency to need badly in current medical R&D process, this exploitation tool for drug development particularly disease of brain medicine is of great significance.
Summary of the invention
In view of the limitation of above-mentioned a series of blood-brain barrier drug kinetic measurement technology, the invention provides a kind of blood brain barrier pharmacokinetics to continue medication system and detection system, particularly relate to a kind of sustained delivery device under animal waking state and continue to extract the dynamic (dynamical) system of real-time detection blood-brain barrier drug that cerebrospinal fluid carries out HPLC analysis, cerebrospinal fluid detection is done by setting up Persistent Csf extraction system and use HPLC under jugular vein continues medication system model and waking state, reach set up a kind of detection of drugs can by blood brain barrier and the assay method by degree.The dynamic (dynamical) Dynamic System of detection blood-brain barrier drug provided by the present invention is simple, and detection accuracy is high, avoids the misery continuing medication and cause to animal, and its application is of great significance zoopery science tool.
Under a kind of animal waking state of the present invention, blood-brain barrier drug kinetic measurement system comprises Persistent Csf extraction system and HPLC cerebrospinal fluid detection system under jugular vein continues medication system, laboratory animal waking state.
First aspect of the present invention there is provided simple, easy and simple to handle and with low cost the continuing medication system through jugular vein of a kind of structure.
What the present invention set up to continue medication system through jugular vein, comprises dosed portions, coupling part.Wherein dosed portions comprises pipe, nut in calparine cap, screw, rigid conduit, injection.Calparine cap, is connected to rigid conduit vertical portion end, prevents leakage and aeroembolism.Screw, upper end can screw with calparine cap, prevents leakage; Lower end can screw with nut, thus in fixing injection, pipe is connected with fibre conduit.Rigid conduit, L-shaped bending, point vertical portion and horizontal part, vertical portion exterior upper portion cover has screw, and can lock with conduit cap and nut, the external diameter of rigid conduit is preferably 0.56mm ± 0.10mm; Pipe in injection, rear connection PE flexible pipe, front end is inserted in rigid conduit, for injectable drug; Nut is positioned at outside screw, for locking pipe and rigid conduit in injection;
Coupling part is made up of PE flexible pipe, and one end of PE flexible pipe is connected with the horizontal part of aforesaid rigid conduit, and the other end is free end; The specification of PE flexible pipe can adjust according to the internal diameter of concrete vessel lumen, is preferably the PE flexible pipe of external diameter 0.85mm, internal diameter 0.42mm; The length of PE flexible pipe also can be determined as the case may be, is preferably 3cm-10cm.
Above-mentioned continue medication system through jugular vein can also comprise pad, coherent mass, Epon, hemostatic clamp and toe-in, its Intermediate gasket is positioned at rigid conduit turning and places perpendicular to the vertical portion of rigid conduit, can play the effect of liquid medicine spill when fixing and prevent intravenously administrable; Described coherent mass is made up of AB glue, and between rigid conduit and pad, effect is bonded on the end face of pad by aforesaid rigid conduit, and aforesaid vertical portion then vertical pad is arranged; Described Epon (epoxy resin) is the sphere made around hose free end by AB glue, is convenient to fix; Hemostatic clamp, for clamp blood vessels during intravenously administrable, the up spilling of anti-Hemostatic Oral Liquid; After toe-in is mainly used in PE flexible pipe implantable intravascular, the fixing PE flexible pipe of knotting binding and blood vessel.
Above-mentionedly be: in body after the jugular vein distal end of ligation side insert PE flexible pipe and ligation is firm at proximal part, set up jugular vein intubation model through the continue medication operation principle of system of jugular vein; External at rat crown heeling-in calparine cap administration port, port engages with PE flexible pipe intubate before (PE flexible pipe is through subcutaneous), thus realizes from external to jugular long-term multiple dosing.
Correspondingly, present invention also offers a kind of animals administer method utilizing above-mentioned sustained delivery device, it comprises the steps:
(1) anaesthetize: by suitable drug anesthesia laboratory animal, get its dorsal position and fix, the jugular vein of surgical exposure laboratory animal;
(2) jugular vein intubation model makes: right jugular vein proximal part clamped by micro-hemostatic clamp, when angioplerosis, distal end ligation, microforceps carries jugular vein distal end, microscissors is oblique towards heart end opening above jugular vein, the intubate being full of heparin inserts jugular vein to Epon place, and behind the two ends of Epon respectively ligation, jugular vein intubate is fixed in mutual ligation;
(3) system test and administration: the fixed installation of laboratory animal cervical region to continue medication system through vein, slowly promotes the syringe be connected with administration end, judges that whether intubate is unobstructed, if unobstructed, can give medicine to be measured after carrying out the stitching of cervical region and head.
Second aspect of the present invention is to provide lasting draw-out device and the method thereof of cerebrospinal fluid under a kind of waking state, this Persistent Csf draw-out device uses glass electrode as syringe needle, puncture the extensive region pillow fascia of laboratory animal, continue to extract cerebrospinal fluid in cerebellomedullary cistern under laboratory animal waking state can be realized.
The Persistent Csf draw-out device that the present invention sets up is by drawing pin formula intubate, coupling part and standing part three ingredients; Wherein drawing pin formula intubate is made up of thin glass syringe needle, PE flexible pipe, medical transparent dressing and 502 glue; PE flexible pipe front end is inserted in thin glass syringe needle, and fixes with 502 glue; Medical transparent dressing is positioned at the front end of drawing pin formula intubate, and the relative position of itself and drawing pin formula intubate can control drawing pin formula intubate and enter the degree of depth that fascia is rested the head in extensive region; Also glue with 502 between intubate and medical transparent dressing, firmly high resilience, fascia can be rested the head on along extensive region and paste steady.
Wherein coupling part mainly contains PE flexible pipe and one-way valve composition.PE flexible pipe mainly plays interconnect function, and the front end of PE flexible pipe is connected with thin glass syringe needle, and rear end continues, and its rear end extendible portion can external microsyringe, and microsyringe stretches can form vacuum drawn cerebrospinal fluid.One-way valve mainly plays single-way switch effect, opens when microsyringe syringe needle inserts, and closes, prevent the backflow of cerebrospinal fluid during cerebrospinal fluid extraction when extracting.The existence of one-way valve can realize extracting cerebrospinal fluid at any time, and cerebrospinal fluid can be avoided again to spill.
Wherein standing part comprises coherent mass and substrate composition, and wherein coherent mass is the coagulated mass that tissue fluid formation is met by tissue glue, can fix PE flexible pipe and make complex stand on thereon.
In above-mentioned lasting extraction cerebrospinal fluid device, material therefor all can experimentally need to select, when extracting cerebrospinal fluid of rats, consumptive material used and specification are preferably: it is most advanced and sophisticated that described thin glass tube is preferably thin glass electrode, superfine, ramped shaped, therefore it rests the head on the minimum and few leakage of epifascial opening in extensive region.Thin glass needle length is 4-5mm; PE flexible pipe preferably forms PE resin by monomer ethylene polymerization and forms, and pipe external diameter is preferably 0.85mm, internal diameter is preferably 0.42mm for it; Gap 2mm*10mm between the hole of described medical air-permeable dressing; Described coherent mass is polyethylene material, basal diameter 0.85cm, high 1.15cm; The same coherent mass of material of described substrate, 0.95cm*2.15cm; The same coherent mass of material of described unidirectional lobe, is polymerized by monomer ethylene, pipe external diameter 1.00mm, internal diameter 0.60mm.
Accordingly, present invention also offers a kind of method utilizing above-mentioned lasting extraction cerebrospinal fluid device extraction animal brain spinal fluid, it is characterized in that comprising the steps:
(1) anaesthetize: by suitable drug anesthesia laboratory animal, get its dorsal position and fix, determine cerebellomedullary cistern approximate location, after exposing extensive region pillow fascia, use distilled water flushing wound surface, aseptic cotton balls is cleaned;
(2) fixing and test: by drawing pin formula intubate by being pressed on cerebral dura mater, tissue glue smears fixing, extracts a small amount of cerebrospinal fluid with microsyringe whether successful to detect intubate;
(3) regularly slowly cerebrospinal fluid is extracted with micro-sampling rifle.
Above-mentioned Persistent Csf draw-out device extracts in the method for laboratory animal cerebrospinal fluid, and experimental implementation operates under Direct Microsurgical, and success rate is higher.
Lasting extraction cerebrospinal fluid device of the present invention, compared with prior art has following beyond thought technical advantage:
A) lasting extraction cerebrospinal fluid device of the present invention pumps cerebrospinal fluid under rat waking state, and the amount pumped is many compared with microdialysis, is also not limited to the small-molecule substance of local cells interstitial fluid, can reflect whole IC situation.
B) the present invention's lasting extraction cerebrospinal fluid apparatus structure is simple, with low cost, making is simple, closure is good, can popularize use.And utilize the present invention continue to extract cerebrospinal fluid device can under waking state at any time whatever you like pump the analysis of animal brain spinal fluid, ensure that medicine is by the verity of blood brain barrier result and accuracy to a greater degree
C) tissue glue that the present invention continues to extract cerebrospinal fluid device used is a kind of tissue adhesive, and be made up of monomer n-butyl-2-acrylic acid cyanogen, this binding substances intensity is high, can rapid polymerization reaction take place after contact tissue liquid.Take out stitches, without the need to using local anaesthesia medicine without the need to seam in experimentation; Few to the local damage of laboratory animal, thus effectively can prevent bacteriological infection, adhesive layer waterproof, anti-pollution; Nontoxic.PE flexible pipe and switch are silicone rubber material in addition.Above-mentioned material all can reduce infection to greatest extent, and ensures the accuracy that final result measures.
D) compared with prior art, the present invention be advantageous in that: the present invention continues to extract cerebrospinal fluid device can get cerebrospinal fluid under rat waking state.Draw-out device structure is simple, and it is convenient to manufacture, and material is easy to get, and manufacturing cost is lower, is convenient to promote the use of, and meets " 3R " principle followed in the world.
3rd aspect of the present invention provides a kind of HPLC cerebrospinal fluid detection system for the above-mentioned state of the art, mainly HPLC analysis is carried out to the cerebrospinal fluid of lasting extraction laboratory animal, namely utilize the extraction cerebrospinal fluid method skillfully grasped repeatedly to extract non-blood contained cerebrospinal fluid and use high performance liquid chromatography to detect in cerebrospinal fluid the medicine and content concn thereof that whether screen containing band, with evaluate this agent permeates therethrough blood brain barrier concrete condition.
Utilize HPLC cerebrospinal fluid detection system to detect a method for cerebrospinal fluid drug content, it comprises the following steps:
A) cerebrospinal fluid sampling: utilize described lasting extraction cerebrospinal fluid apparatus system to extract certain volume animal brain spinal fluid at set intervals.Further, the present inventor finds, generally extracts a cerebrospinal fluid every 5 minutes and can meet the content situation of reflection medicine in cerebrospinal fluid.
B) cerebrospinal fluid process: contain other materials a large amount of in cerebrospinal fluid as high molecular weight protein, the accuracy that high-efficient liquid Relative drug detects can be affected.Therefore, in cerebrospinal fluid processing procedure, the centrifugal removing high molecular weight protein of cerebrospinal fluid High speed refrigerated centrifuge obtained will be extracted.
C) efficient Liquid Detection: utilize the concentration of efficient liquid phase conventional means detection of drugs in cerebrospinal fluid, and in conjunction with administrations to Analysis of test results, reach a conclusion.
Present invention also offers and a kind ofly utilize under animal waking state described above that blood-brain barrier drug kinetic measurement system measurement medicine is by the method for blood brain barrier, it mainly comprises the steps:
A) by laboratory animal with after suitable drug anesthesia, install and to continue medication sustained delivery device through jugular vein, give medicine to be measured;
B) install after jugular vein sustained delivery device, can realize repeatedly giving medicine to be measured through jugular vein in effective time span;
C) after administration certain hour, laboratory animal is installed Persistent Csf draw-out device, under waking state, extract cerebrospinal fluid by certain frequency in laboratory animal cerebellomedullary cistern;
D) by cerebrospinal fluid frozen centrifugation removing large protein, high performance liquid chromatography is utilized to detect cerebrospinal fluid drug concentration.
Said determination medicine, by the method for blood brain barrier, goes for common laboratory animal, and as rat, mice, cat, dog, rabbit, duck, pig, monkey etc., being preferably applicable to take rat as the situation of modeling zoometry medicine by blood brain barrier.
Under animal waking state described above, blood-brain barrier drug kinetic measurement system measurement medicine is by the method for blood brain barrier, and the embodiment of its best is as described below:
A) laboratory animal anesthesia: by suitable drug anesthesia laboratory animal, get its dorsal position and fix;
B) jugular vein intubation model makes: the jugular vein of surgical exposure laboratory animal, right jugular vein proximal part clamped by micro-hemostatic clamp, when angioplerosis, distal end ligation, microforceps carries jugular vein distal end, microscissors is tiltedly towards heart end opening above jugular vein, and the intubate being full of heparin inserts jugular vein to Epon place, and behind the two ends of Epon respectively ligation, jugular vein intubate is fixed in mutual ligation;
C) fix through the vein system that continues medication, test and administration: laboratory animal cervical fixation systems, slowly promotes the syringe be connected with administration end, judge that whether intubate is unobstructed, if unobstructed, can give medicine to be measured after carrying out the stitching of cervical region and head.
D) continue cerebrospinal fluid extraction system fix and test: determine cerebellomedullary cistern approximate location, after exposing extensive region pillow fascia, by drawing pin formula intubate by being pressed on cerebral dura mater, tissue glue smears fixing, extracts a small amount of cerebrospinal fluid whether successful to detect intubate with microsyringe.
E) cerebrospinal fluid extraction and process: regularly slowly extract cerebrospinal fluid with micro-sampling rifle, to be measured after High speed refrigerated centrifuge centrifugal removing high molecular weight protein.
F) cerebrospinal fluid Chinese medicine detects: the content that has that it's too late utilizing medicine to be measured in high performance liquid chromatography determination cerebrospinal fluid.
Under animal waking state of the present invention, blood-brain barrier drug kinetic measurement system is compared with prior art, at least achieves following outstanding unexpected technique effect:
1. the intravenous injection difficulty of laboratory animal particularly rat or mice is large, and continues medication and bring many miseries to laboratory animal, and the present invention solves the difficult problem continued medication of laboratory animal by employing jugular vein intubation technique, and simple to operate;
2. the present invention is to the improvement of conventional vein intubation technique, solves the problem owing to easily causing intubate to come off during laboratory animal own activity;
3. the improvement of surgical technic, makes laboratory animal jugular vein intubation model survival rate greatly improve;
4. the present invention uses glass electrode to extract cerebrospinal fluid as syringe needle, and controls syringe needle penetration depth with medical dressing, effectively solves because inserting needle causes the problem of laboratory animal death excessively deeply;
5. the present invention use glass electrode because of its syringe needle thin, solve because repeating to extract the problem causing extensive region to rest the head on fascial rupture;
6. operate all under the microscope when detection system of the present invention uses, solve the problem of the cerebrospinal fluid band blood of extraction, thus improve medicine by the accuracy of blood brain barrier testing result and reliability.
Accompanying drawing explanation
Fig. 1 is the system schematic that continues medication through jugular vein;
Fig. 2 continues to extract cerebrospinal fluid apparatus structure schematic diagram;
Fig. 3 continues the structural representation extracting cerebrospinal fluid device standing part;
Fig. 4 continues to extract cerebrospinal fluid device cannula structures schematic diagram;
Fig. 5 continues to extract cerebrospinal fluid device switch structure diagram;
Fig. 6 rat inserts extensive region pillow fascia analogous diagram.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is further described in detail.It should be appreciated by those skilled in the art that embodiment and do not limit the present invention in any way, any do on basis of the present invention equivalent to substitute all within the scope of protection of present invention.
Embodiment 1 illustrated blood brain barrier kinetic measurement system
Fig. 1 shows the optimal way of a kind of the best of blood brain barrier kinetic measurement system under waking state of the present invention to 6.
As shown in Figure 1, under a kind of laboratory animal waking state that the present invention sets up, blood brain barrier pharmacokinetics continues medication system, comprises dosed portions, coupling part and standing part.Wherein dosed portions comprises pipe 5, nut 6 in calparine cap 1, screw 2, rigid conduit 3, injection.Calparine cap 1, is connected to rigid conduit vertical portion end, can prevents leakage and aeroembolism.Screw 2, upper end can screw with calparine cap 1, and lower end can screw with nut 6, thus in fixing injection, pipe is connected with fibre conduit.Rigid conduit 3, L-shaped bending, point vertical portion and horizontal part, top, vertical portion overcoat has screw 2, and can lock with calparine cap 1 and nut 6, the external diameter of rigid conduit 3 is preferably 0.56mm ± 0.10mm; Injection in pipe 5, after connect PE flexible pipe, front end is inserted in rigid conduit, for injectable drug; Nut 6, for locking pipe and rigid conduit in injection;
Coupling part is made up of PE flexible pipe 7, and one end of PE flexible pipe 7 is connected with the horizontal part of aforementioned rigid conduit 3, and the other end is free end; When administration, free end can be inserted in the blood vessel of animal.The specification of above-mentioned PE flexible pipe 7 can adjust according to the internal diameter of concrete vessel lumen, is preferably the PE flexible pipe of external diameter 0.85mm, internal diameter 0.42mm; The length of PE flexible pipe also can be determined as the case may be, is preferably 3cm-10cm.
Above-mentioned continue medication system through jugular vein can also comprise pad 4, coherent mass, Epon 8, hemostatic clamp 9 and toe-in 10, its Intermediate gasket 4 is positioned at rigid conduit turning and places perpendicular to the vertical portion of rigid conduit, can play the effect of liquid medicine spill when fixing and prevent intravenously administrable; Described coherent mass is made up of AB glue, and between rigid conduit and pad, effect is bonded on the end face of pad by aforesaid rigid conduit, and aforesaid vertical portion then vertical pad is arranged; Described Epon 8 (epoxy resin) is the sphere made around hose free end by AB glue, is convenient to fix; Hemostatic clamp 9, for clamp blood vessels during intravenously administrable, the up spilling of anti-Hemostatic Oral Liquid; After toe-in 10 is mainly used in PE flexible pipe implantable intravascular, the fixing PE flexible pipe of knotting binding and blood vessel.
The operation principle of above-mentioned drug-supplying system is: elongated foremost by PE flexible pipe before administration, carry out Epon at about 2-3mm place AB glue.After hemostatic clamp 9 in vivo ligation side jugular vein distal end, insert the free end of PE pipe 7 at proximal part, and firm by toe-in 10 ligation.External at rat crown heeling-in calparine cap administration port, wherein body the inner one immediately PE pipe (namely 7, through subcutaneous) of part in body, thus test and be administered to jugular vein in vitro according to certain injection rate.
Accompanying drawing 2 is to fig. 5 illustrating blood-brain barrier drug kinetic measurement system Persistent Csf draw-out device part under animal waking state, wherein as shown in Figure 2, described Persistent Csf draw-out device is by drawing pin formula intubate, coupling part and standing part three ingredients;
Wherein, as Fig. 4 continues to extract shown in cerebrospinal fluid device cannula structures schematic diagram, drawing pin formula intubate is made up of thin glass syringe needle 5, PE flexible pipe 8, medical transparent dressing 6 and 502 glue 4; PE flexible pipe 8 front end is inserted in thin glass syringe needle, and fixes with 502 glue; Medical transparent dressing is positioned at the front end of drawing pin formula intubate, and the relative position of itself and drawing pin formula intubate can control drawing pin formula intubate and enter the degree of depth that fascia is rested the head in extensive region; Also glue with 502 between intubate and medical transparent dressing, firmly high resilience, fascia can be rested the head on along extensive region and paste steady.Tissue glue meets tissue fluid and solidifies immediately, and viscosity further enhances well the steadiness of intubate.For when ensureing end tractive further, intubate does not move, flexible pipe up pastes along occipital bone with dressing and tissue fluid by we always.
Wherein coupling part mainly contains PE flexible pipe 1 and one-way valve 3 forms.PE flexible pipe mainly plays interconnect function, and the front end of PE flexible pipe is connected with thin glass syringe needle, and rear end continues, and its rear end extendible portion can external microsyringe, and microsyringe stretches can form vacuum drawn cerebrospinal fluid.One-way valve mainly plays single-way switch effect, opens when microsyringe syringe needle inserts, and closes, prevent the backflow of cerebrospinal fluid during cerebrospinal fluid extraction when extracting.The existence of one-way valve can realize extracting cerebrospinal fluid at any time, and cerebrospinal fluid can be avoided again to spill.
Wherein standing part comprises coherent mass 9 and substrate 7 forms, and wherein coherent mass is the coagulated mass that tissue glue meets tissue fluid and formed, and can fix PE flexible pipe and complex is stood on above substrate 7.
Fig. 3 of the present invention shows the structural representation continuing to extract cerebrospinal fluid device standing part, wherein one-way valve 3 is arranged in PE flexible pipe 2, mainly play single-way switch effect, open when microsyringe syringe needle inserts, close when extracting, prevent the backflow of cerebrospinal fluid during cerebrospinal fluid extraction, the outside of PE flexible pipe 2 is wrapped to form complex by 502 glue, this complex is connected with substrate 7 in substrate accompanying drawing 1, is convenient to the fixing of draw-out device.
The present invention is attached Figure 6 shows that rat inserts extensive region pillow fascia analogous diagram, this analogous diagram shows the fixed position of Persistent Csf draw-out device, wherein fascia worn out by the thin glass syringe needle 1 of drawing pin formula intubate, medical transparent dressing 2 to be bonded on fascia fixing, and the relative position of itself and drawing pin formula intubate can control the penetration depth of drawing pin formula intubate.PE flexible pipe 4 being posted along occipital bone, with organizing glue 3, drawing pin formula intubate being fixed further.
The HPLC cerebrospinal fluid detection system that the present invention sets up is the conventional means of this area, does not repeat at this.
Under utilizing above-mentioned laboratory animal waking state, blood brain barrier pharmacokinetics Analytical system measures the method for medicine by blood brain barrier, and it comprises following steps:
A) laboratory animal anesthesia: by suitable drug anesthesia laboratory animal, get its dorsal position and fix;
B) jugular vein intubation model makes: the jugular vein of surgical exposure laboratory animal, right jugular vein proximal part clamped by micro-hemostatic clamp, when angioplerosis, distal end ligation, microforceps carries jugular vein distal end, microscissors is tiltedly towards heart end opening above jugular vein, and the intubate being full of heparin inserts jugular vein to Epon place, and behind the two ends of Epon respectively ligation, jugular vein intubate is fixed in mutual ligation;
C) fix through the vein system that continues medication, test and administration: laboratory animal cervical fixation systems, slowly promotes the syringe be connected with administration end, judge that whether intubate is unobstructed, if unobstructed, can give medicine to be measured after carrying out the stitching of cervical region and head.
D) continue cerebrospinal fluid extraction system fix and test: determine cerebellomedullary cistern approximate location, after exposing extensive region pillow fascia, by drawing pin formula intubate by being pressed on cerebral dura mater, tissue glue smears fixing, extracts a small amount of cerebrospinal fluid whether successful to detect intubate with microsyringe.
E) cerebrospinal fluid extraction and process: regularly slowly extract cerebrospinal fluid with micro-sampling rifle, to be measured after High speed refrigerated centrifuge centrifugal removing high molecular weight protein.
F) cerebrospinal fluid Chinese medicine detects: the content that has that it's too late utilizing medicine to be measured in high performance liquid chromatography determination cerebrospinal fluid.
Embodiment two utilizes illustrated detection system research heparin by blood brain barrier of rats situation
1, utilizing continues medication through vein is administered systemically
1. 10% chloral hydrate 0.3ml/100g urethane (1.2g/kg) anesthetized rat, gets dorsal position and fixes; Eye is sealed with chlortetracycline eye ointment, at front tooth---right axillary fossa and auris dextra before---intersection area of left axillary fossa line delimited the first operative region and carried out first time preserved skin, in the first operation locality disinfection, lay operation towel with holes, be cut into longitudinal skin incision at the first operative region, about 1-1.5cm, cut subcutaneous depth fascia, process of stopping blooding in time, along spatium intermusculare along machine direction blunt separation subcutaneous tissue and muscular tissue, appear and the right jugular vein that dissociates, thereunder threading ligation is for subsequent use;
2. right jugular vein proximal part clamped by micro-hemostatic clamp, when angioplerosis, distal end ligation, microforceps carries jugular vein distal end, microscissors is oblique towards heart end opening above jugular vein, the intubate being full of heparin inserts jugular vein to Epon place, and behind the two ends of Epon respectively ligation, jugular vein intubate is fixed in mutual ligation, and intubate total length is about 3.5-4.5cm;
3. on the skull platform of rat two ear line precontract 1cm, open the opening of about 0.8-1.5cm, the other end of micro-mosquito forceps clamping intubate, head opening part is arrived walk around ear from cervical region is subcutaneous after, it is connected with the head piece being full of heparin, under head piece is embedded in scalp, slowly promotes the syringe be connected with administration end, judge that whether intubate is unobstructed, if unobstructed, carry out the stitching of cervical region and head, smear chlortetracycline eye ointment at suture.
By pre-configured heparin solution (heparin concentration is that 20%, 100mg heparin is dissolved in 0.9% normal saline 400mL) through above-mentioned through the administration of jugular vein drug-supplying system, after administration, 20min starts to extract cerebrospinal fluid.
2, utilize the present invention to continue to extract cerebrospinal fluid device and extract cerebrospinal fluid of rats
1. the 10% chloral hydrate anesthesia rat of lumbar injection 0.3ml/100g.Determine cerebellomedullary cistern approximate location with hands, clipper for surgical use cuts off surperficial hair.Utilize Naoliqing capsule ear bar, front tooth bar fixes rat head.A stringer otch (about 1.5-2.5cm) is cut, with musculi colli, tissue after mosquito forceps and tweezers blunt separation under micrurgy along posterior midline cerebellomedullary cistern body surface projection.The muscle operation knife back that bottommost layer is attached to occipital bone L scrapes off, hemorrhage to avoid, and after exposing extensive region pillow fascia, use distilled water flushing wound surface, aseptic cotton balls is cleaned.
2. drawing pin formula intubate be connected with single-way switch by flexible pipe, surgical glue smears drawing pin surrounding, and under surgical microscope, by drawing pin by being pressed on cerebral dura mater, fixing intubate is smeared by tissue glue.A small amount of cerebrospinal fluid is extracted whether successful to detect intubate with microsyringe.Be adjacent to fixing along occipital bone by flexible pipe, suture muscles and opening, switch partly buries with subcutaneous.To rat injection penicillin.
3. regularly cerebrospinal fluid 20ul is slowly extracted with micro-sampling rifle.Time gradient is preferably every 5 minutes and extracts a cerebrospinal fluid, extracts 5 times or more number of times altogether.
3, high performance liquid chromatography detects cerebrospinal fluid drug concentration
3.1 cerebrospinal fluid pre-treatments
Get cerebrospinal fluid 20 microlitre with micro-sampling rifle by the lasting extraction cerebrospinal fluid device in the present invention, add 0.4mol/L and cross chloric acid (PCA) 180 microlitre, mixing, 37 DEG C, the centrifugal 10min of 12000rmin.Get supernatant 20 microlitre and be diluted to 500 microlitres.
3.2 high performance liquid chromatography detect cerebrospinal fluid drug concentration experimental procedure
1) the HPLC flow visualizing being suitable for substance A adopts ODS post, take acetonitrile/water as flow phase system.By regulating the pH value of phosphate/acetonitrile system, make the interference Component seperation in target oligopeptide and cerebrospinal fluid and blood plasma, whether object observing peak occurs.
2) stability of A material in tissue fluid is according to substance A appearance time and pH system, get No. 1,5 different time samples, No. 2, No. 3, No. 4, No. 5, by setting interval get 10 microlitersasample solution inject HPLC
Analyze and discuss: after self administration of medication, 20min rises, a spinal fluid samples is got every 5min, high-performance liquid chromatogram determination is entered after spinal fluid samples process, testing result shows, do not find the existence of heparin in administration 20min-45min in cerebrospinal fluid, point out heparin at 20min-45min not by blood brain barrier.
Embodiment three utilizes illustrated detection system research maize oligopeptide, Semen Tritici aestivi oligopeptide by blood brain barrier of rats situation
Laboratory operating procedures and modelling are with embodiment one, be maize oligopeptide, Semen Tritici aestivi oligopeptide unlike administration, be made into 0.01mg/ml solution, every Mus injection 0.5ml, result shows, after administration 20min, show maize oligopeptide in cerebrospinal fluid, Semen Tritici aestivi oligopeptide exists, and in 20min-45min, maize oligopeptide, the Semen Tritici aestivi oligopeptide cerebrospinal fluid drug concentration when 40min is the highest, points out this medicine easily to pass through blood brain barrier.
Claims (1)
1. a blood-brain barrier drug kinetic measurement system under animal waking state, is characterized in that, it comprises Persistent Csf extraction system and HPLC cerebrospinal fluid detection system under jugular vein continues medication system, laboratory animal waking state;
Described comprise dosed portions, coupling part through the jugular vein system that continues medication;
Wherein dosed portions comprises pipe, nut in calparine cap, screw, rigid conduit, injection; Calparine cap, is connected to rigid conduit vertical portion end, prevents leakage and aeroembolism; Screw, upper end can screw with calparine cap, prevents leakage; Lower end can screw with nut, thus manages and rigid tubes connection in fixing injection; Rigid conduit, L-shaped bending, point vertical portion and horizontal part, vertical portion exterior upper portion cover has screw, can lock with calparine cap and nut, and the external diameter of rigid conduit is 0.56mm ± 0.10mm; Pipe in injection, rear connection PE flexible pipe, front end is inserted in rigid conduit, for injectable drug; Nut is positioned at outside screw, for locking pipe and rigid conduit in injection;
Wherein coupling part is made up of PE flexible pipe, and one end of PE flexible pipe is connected with the horizontal part of aforesaid rigid conduit, and the other end is free end;
Described can also comprise pad, coherent mass, Epon (epoxy resin), hemostatic clamp and toe-in through the jugular vein system that continues medication; Its Intermediate gasket is positioned at rigid conduit turning and places perpendicular to the vertical portion of rigid conduit; Described coherent mass is made up of AB glue, and between rigid conduit and pad, aforesaid vertical portion then vertical pad is arranged; Described Epon (epoxy resin) is the sphere made around hose free end by AB glue, is convenient to fix; Hemostatic clamp, for clamp blood vessels during intravenously administrable; After toe-in is mainly used in PE flexible pipe implantable intravascular, the fixing PE flexible pipe of knotting binding and blood vessel;
Under described laboratory animal waking state, Persistent Csf extraction system is made up of drawing pin formula intubate, coupling part and standing part;
Wherein drawing pin formula intubate is made up of thin glass syringe needle, PE flexible pipe, medical transparent dressing and 502 glue; PE flexible pipe front end is inserted in thin glass syringe needle, and fixes with 502 glue; Medical transparent dressing is positioned at the front end of drawing pin formula intubate, and its relative position in drawing pin formula intubate can control drawing pin formula intubate; Front end can be inserted and is fixed on the pillow fascia of extensive region, and it controls by medical transparent dressing;
Wherein coupling part mainly contains PE flexible pipe and one-way valve composition; PE flexible pipe mainly plays interconnect function, and one-way valve mainly plays single-way switch effect, opens when microsyringe syringe needle inserts, and closes, prevent the backflow of cerebrospinal fluid during cerebrospinal fluid extraction when extracting; The outside of PE flexible pipe is wrapped to form complex by 502 glue; The existence of one-way valve can realize extracting cerebrospinal fluid at any time, and cerebrospinal fluid can be avoided again to spill;
Wherein standing part comprises coherent mass and substrate composition, and wherein coherent mass is the coagulated mass that tissue fluid formation is met by tissue glue, mainly plays fixing PE flexible pipe and makes complex stand on thereon.
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| CN104546211B (en) * | 2013-10-29 | 2018-04-20 | 南京中医药大学 | A kind of miniature jugular vein of rat is administered and takes blood system |
| CN103598924B (en) * | 2013-12-03 | 2015-09-09 | 菏泽学院 | Experimental rat nervus centralis draws drawing device |
| CN104323868A (en) * | 2014-11-06 | 2015-02-04 | 中国人民解放军第四军医大学 | Micro-infusion method and device thereof based on epididymis duct intracavity environment experiment |
| CN104874093A (en) * | 2015-04-24 | 2015-09-02 | 中山大学 | Cerebrospinal fluid extracting device |
| CN108670491A (en) * | 2018-06-11 | 2018-10-19 | 新乡医学院 | A kind of mouse cerebrospinal fluid acquisition system and acquisition method |
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