Summary of the invention
In view of the deficiency that background technology exists, technical problem to be solved by this invention be to provide a kind of simple to operate, test short and experimental technique to blood vessel 3D structure in the inert Reconstruction of Rabbit terminal plate of vertebral body of experimenter consuming time.
The present invention takes following technical scheme: the experimental technique of blood vessel 3D structure in Reconstruction of Rabbit terminal plate of vertebral body, is characterized in that: comprising:
1) prepare contrast agent: be dissolved in by 48g barium sulfate in normal saline and the pure water solution of 80ml equal proportion mixing, mixing and being placed on power is in the sonicator of about 25W about 5 minutes, then it is stand-by to put into about 50 DEG C of water baths;
2) injection of contrast medium
The method that experimental rabbit adopts 10% chloral hydrate (1.5ml/kg) auricular vein to inject gives to implement general anesthesia; After determining that anesthesia reaches certain depth, set laboratory animal operation position, deduct abdomen area hair; Abdomen median line cuts abdominal cavity, exposes renal artery with lower abdominal aorta, postcava and bilateral common iliac artery; Silk thread ligation bilateral common iliac artery, carry out intubate to ventral aorta subsequently and pour into heparin-saline (500u/100ml), inferior vena cava cannula gives blood-letting, and heparin-saline rinses blood vessel until effluent is refrigerant; Backward ventral aorta in injection of contrast medium (about 20ml/min) until postcava in flow out milky white liquid (about 60ml); After injection, close ventral aorta, postcava;
3) MicroCT scanning
Draw materials after 1h: often only take whole L4-5 vertebral body, adopt U.S. PerkinElmer company QuantumFX toy CT system to carry out specimen scanning; Selection sweep parameter is as follows: model: QuantumFX, voltage: 90KV, electric current: 160UA; Same organizing first scans 10mm range size, then scans soleplate central area 5mm range size; 10MM group and 5MM group is divided into by scanning the data obtained;
4) three-dimensional reconstruction
The scan-data obtained is converted into DICOM formatted file and imports Mimics software (15.0), set up soleplate according to sclerotin respectively from the different threshold size of Hacebar and remove the blood vessel 3D after soleplate sclerotin and scheme, random selecting 5 blood vessel ends from the blood vessel 3D figure of two groups of experimental results respectively, measure its diameter, average and represent tail vein diameter.
In the present invention, MicroCT is used to scan the rabbit soleplate after injection Hacebar, and use Mimics software to carry out 3D to scanning result to rebuild the vascular bud morphological structure of observing in rabbit soleplate, experiment is had simple to operate, consuming time short and without advantages such as hazardness, and observable blood vessel three dimensional structure is high-visible, is more easy to observation and analysis.
Detailed description of the invention
Drawings illustrate technical scheme of the present invention and embodiment, further describe each details and the operation principle thereof of embodiment below more by reference to the accompanying drawings.
With reference to shown in Fig. 1, Fig. 2, Fig. 3, Fig. 4, Fig. 5, Fig. 6, the experimental technique of blood vessel 3D structure in Reconstruction of Rabbit terminal plate of vertebral body provided by the invention, comprising:
One, experiment material is selected:
6 monthly age Japan large ear rabbits (2.5 ~ 3kg) 5, Wenzhou Medical University's animal experimental center provides, product batch number: SYXK (Zhejiang) 2014-0006; Shanghai ten thousand boundary barium sulfate (specification: 99%, 1 μm); PerkinElmerQuantumFX toy CT system; U.S. Sonics sonicator.
Two, experimental technique
1) contrast agent prepares
Be dissolved in by 48g barium sulfate in normal saline and the pure water solution of 80ml equal proportion mixing, mixing and being placed on power is in the sonicator of about 25W about 5 minutes, then it is stand-by to put into about 50 DEG C of water baths.
2) injection of contrast medium
The method that experimental rabbit adopts 10% chloral hydrate (1.5ml/kg) auricular vein to inject gives to implement general anesthesia.Determine anesthesia reach certain depth after (its extremity of vascular forceps clamp react without muscle contraction, without corneal reflex) set laboratory animal operation position (i.e. dorsal position), deduct abdomen area hair.Abdomen median line cuts abdominal cavity, exposes renal artery with lower abdominal aorta, postcava and bilateral common iliac artery.Silk thread ligation bilateral common iliac artery, carry out intubate to ventral aorta subsequently and pour into heparin-saline (500u/100ml), inferior vena cava cannula gives blood-letting, and heparin-saline rinses blood vessel until effluent is refrigerant.Backward ventral aorta in injection of contrast medium (about 20ml/min) until postcava in flow out milky white liquid (about 60ml).After injection, close ventral aorta, postcava.
3) MicroCT scanning
Draw materials after 1h: often only take whole L4-5 vertebral body, adopt U.S. PerkinElmer company QuantumFX toy CT system to carry out specimen scanning.Selection sweep parameter is as follows: model: QuantumFX, voltage: 90KV, electric current: 160UA.Same organizing first scans 10mm range size, then scans soleplate central area 5mm range size.10MM group and 5MM group is divided into by scanning the data obtained.
4) three-dimensional reconstruction
The scan-data obtained is converted into DICOM formatted file and imports Mimics software (15.0).Set up soleplate according to sclerotin respectively from the different threshold size of Hacebar and remove the blood vessel 3D after soleplate sclerotin and scheme.Random selecting 5 blood vessel ends from the blood vessel 3D figure of two groups of experimental results, measure its diameter, average and represent tail vein diameter respectively.
Statistical analysis:
SPSS19.0 statistics software is adopted to carry out statistical analysis.The data obtained adopts t inspection to carry out statistical procedures; Data with mean ± standard deviation (± s) represent, two-sided test, α=0.05,p< 0.05 has significance for difference.
Three, result
Result 1.MicroCT scanning result is observed: application Mimics software obtains the shaft-like position of terminal plate of vertebral body and vertebral body sclerotin on L4, Coronal and sagittal image, wherein can obviously observe soleplate structure, there is obvious watershed area and growth plate position in the vertebral body that soleplate is adjacent.There is the pore passage structure of volume in soleplate inside, each pore passage structure dependent exist, and have pathways mutually between contiguous duct.Barium sulfate angiography is obviously developed in the pore passage structure in soleplate, and in shaft-like bit image, we find soleplate internal blood vessel bud through hole structure, and complexity out of shape, main integrated distribution is in middle section (Fig. 1, Fig. 2).
Result 2.Mimics three-dimensional reconstruction result is observed: the visualisation in terminal plate of vertebral body is carried out 3D reconstruction, obtains its 3D rendering, and this image is with reference to Fig. 3.Blood vessel three dimensional structure in terminal plate of vertebral body is high-visible.Soleplate blood vessel 10mm group top view visible vessels is intensive in vertebral pulp site distribution, and caliber is thick, and to the fiber ring portion radially radiation (Fig. 3) of surrounding; On sagittal plane, vertebral body and intervertebral disc are obviously separated by visible soleplate, and in it, blood vessel sends from vertebral body rear, grow into along the forwards radiation of soleplate rear, and intervertebral disc in and have no blood vessel exist (Fig. 4).See in meticulousr 5mm image that the blood vessel of soleplate center nucleus pulposus area is different from the simple button loop spline structure of periphery fiber ring portion blood vessel, the shape of blood vessel not single button loop spline structure of nucleus pulposus portion, but a kind of blood vessel be intertwined completely of bulky complex button loop structure (Fig. 5).
3. tail vein diameters are analyzed as a result: the tail vein diameter measurement result of 10MM group and 5MM group as shown in Figure 6.The tail vein diameter of 10MM group and 5MM group compares analysis, and difference has significant difference (Fig. 6).
The route of nutrition of intervertebral disc cells is divided into two parts, 1. fibrous ring approach: nutrient substance enters fibrous ring skin by the blood vessel being distributed in fibrous ring surface (mainly outer 1/3 annular region), and nutrition scope is little.By the method for being injected by fluorescent dye, Brrodin etc. find that dyestuff mainly concentrates on outer layer fiber ring region, and in vertebral pulp, do not observe the appearance of fluorescent dye.2. soleplate approach (main path): nutrient substance diffuses to intervertebral disc by the osmosis of the blood vessel in soleplate, nutrition inner fiber ring and vertebral pulp.2007, by blocking Ren Caprae seu Ovis intervertebral disc soleplate, vanderwerf etc. showed that blocking soleplate approach can suppress endovascular solute molecule to be transported to nuclear area, in this prompting soleplate, microvascular existence is the important foundation of nutrient substance transhipment in vertebral pulp.And Rodriguez etc. are by observing the degenerative processes of people's lumbar vertebra soleplate topology discovery along with intervertebral disc of regression, soleplate sclerosis is main at external zones, the nucleus pulposus cell of intervertebral disc central area is but along with the sclerosis of soleplate is the trend that cell density increases, and the activity of nucleus pulposus cell obviously reduces.Therefore most important for the exploration of blood vessel structure in soleplate.
As far back as nineteen nineties, Oki etc. scan the vascular morphology observed in soleplate by scanning electron microscope after blood vessel casting.The scholars such as domestic Lv Gang use the method for vascular perfusion ABS also successfully to prepare the blood vessel casting specimen of rabbit lumbar vertebral body cartilage endplate, and pass through the morphological structure of vascular bud in projection light microscope and scanning electron microscopic observation terminal plate of vertebral body.Both all find that in soleplate, blood vessel is dendroid distribution, are woven and form blood vessel button loop spline structure, i.e. vascular bud.These vascular buds are sparse in inner fiber ring site distribution, and vascular bud shape is in simply fastening with a rope, string, etc. spline structure, and vertebral pulp site distribution is intensive, expand and the button loop spline structure that is woven of complexity in a kind of.This position high osmosis may have larger surface area with its blood vessel and contact point is more relevant.But need to use some dangerous goods such as organic solvent and concentrated hydrochloric acid preparing in blood vessel casting specimen process, and specimen preparation procedure needs several days time, process is loaded down with trivial details.And this experiment scans the rabbit soleplate structure after injection of contrast medium by MicroCT, and Mimics system is used to carry out 3D reconstruction to blood vessel structure in soleplate.Wherein contrast agent is clinical conventional barium sulfate, to human non-toxic's side effect, and can scan immediately after having injected, without the need to waiting for.There is advantage simply, safely and fast, and acquired results is consistent with early-stage Study.
In scanning process, we have attempted 10mm and 5mm range scans, there is notable difference between the two for blood vessel precision, and 5mm range scans can reach minimum 20um precision.The average blood vessel diameter of 5mm group reaches 35um, the average caliber of 10mm group is 70um, this is relevant with scanning accuracy, 10mm group and 5mm group are continuous sweep 512 layers, there is different layer distances between the two, and layer is apart from determining scanning accuracy, therefore two group end blood vessel diameter measurement results demonstrate obvious difference.On contrast agent is selected, we have successively attempted the Hacebar of concentration about 150%, 100% and 60%, larger resistance is there is in the above two in injection process, often cause angiorrhexis situation to occur because syringe pressure is excessive, and still there is part injection resistance in 60% concentration in injection process, though can injection process be completed, but it is not light, so this seminar injects after this concentration contrast agent is carried out ultrasonic Treatment again, easily complete injection process with the injection rate of about 20ml/min like this.
This seminar successfully establishes the blood vessel 3D rendering in soleplate by injection Hacebar, blood vessel in discovery terminal plate of vertebral body is interlaced forms blood vessel button loop and vascular bud form in dendroid, and it is densely distributed in nucleus pulposus area, and form is compared with inner fiber ring region more complicated, this morphosis makes this regional vessel have larger surface area and more contact point, improves the permeability of nucleus pulposus area.This experiment first passage the method explores blood vessel structure in soleplate.