CN101915693B - Human embryonic trigeminus based three-dimensional reconstruction method by using slide stainer - Google Patents
Human embryonic trigeminus based three-dimensional reconstruction method by using slide stainer Download PDFInfo
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Abstract
The invention discloses a human embryonic trigeminus based three-dimensional reconstruction method by using histotomy staining. In the method, trigeminal ganglions, roots and branches of a fresh sample of a human embryo, especially an over 20-week induced-labor infant embryo not died with diseases are selected and subjected to HE conventional staining and observed under a light microscope and a transmission electron microscope, and finally the three-dimensional reconstruction is realized by using a computer TRI for win 95/NT, Version:3.01. By researching the form of the human embryonic trigeminus and advancing of motor roots, the invention shows the three-dimensional structure of the human embryonic trigeminus for the first time in human history, which not only enriches the research content of morphology of human embryonic trigeminus, but also provides a morphological basis for clinical prosopalgia pathophysiology and treatment. And thus, the human embryonic trigeminus based three-dimensional reconstruction method by using histotomy staining has a wide application scope.
Description
Technical field
The invention belongs to the neurotomy technical field.Specifically, the present invention relates to a kind ofly utilize tissue section strain to carry out the technical field of three-dimensional reconstruction based on people embryo trigeminal neuralgia.
Background technology
Trigeminal neuralgia (trigeminal nerve) is thick Combination cranial nerve, contains two kinds of fibers of general somatesthesia and special internal organ motion.Special visceral motor fiber arises from the nuclei motorii nervi trigemini in pons stage casing; Its aixs cylinder is formed motor root of trigeminal nerve; Basilar part of pons (from the pons facies ventralis) goes out brain with the medipeduncle place of dividing a word with a hyphen at the end of a line, and is positioned at the preceding inboard of sensory root, gets into mandibular nerve at last; Go out cranium through oval foramen, branch is distributed in masseter etc. with mandibular nerve.The cell space of general somatesthesia fiber concentrates in the gasserian ganglion, and wherein central process is concentrated and constituted thick radix sensoria nervi trigemini, goes into brain by basilar part of pons and medipeduncle intersection, terminates in all sensory nucleus of trigeminal neuralgia; The prominent trigeminal neuralgia three big branches that form around the trigeminal neuralgia ganglion cell, i.e. the 1st ophthalmic nerve, the 2nd maxillary nerve, the 3rd is mandibular nerve.From 3 continuous branches of big branch be distributed in skin of face, eye and the socket of the eye, the mucous membrane of oral cavity, nasal cavity, paranasal sinus, tooth, meninxes etc., polyesthesia such as touch at conduction pain, temperature.
(trigeminal neuralgia is modal a kind of in the cranial neuralgia TN) to trigeminal neuralgia, also is one of disease common in the peripheral nervous disease.According to statistics; This sick morbidity rate is 1,82/,100,000; Morbidity is a characteristic with the lightening violent facial pain that takes place suddenly in one of facial trigeminal neuralgia or several distributive province, and the patient often is described as and tears appearance, get an electric shock appearance, lightning appearance, acupuncture appearance, lancinates kind or burn a kind severe pain.Pain is the most obvious with cheek, the upper jaw, lower jaw or tongue.Because the definite pathogenesis of trigeminal neuralgia and the cause of disease it be unclear that, thus desirable methods of treatment lacked clinically, common with surgical operation therapy.The selectivity sensory rhizotomy of trigeminal nerve is one of surgical intervention trigeminal neuralgia method commonly used at present.But can damage motor root sometimes in the art, show as the homonymy masticatory paralysis, dental articulation is not tight, and some patient Yi Fasheng temporomandibular joint dislocation also has some patients difficulty in opening mouth can occur, and lower jaw is partial to Ipsilateral etc. when dehiscing.Need differentiate sensory branches and the relation of moving and propping up in the gasserian ganglion in the art.Therefore, research, the especially motor root of trigeminal nerve traveling in gasserian ganglion of Roots of trigeminal nerve morphology aspect has certain theory and practice significance to the prosopalgic cause of disease and treatment.
The traveling of form and the motor root of trigeminal nerve of adult's Roots of trigeminal nerve has been received the concern of many Chinese scholars, and this has been carried out a series of research.People such as Young find in limited time that in the motion of research adult trigeminal neuralgia motor root contains the Remak's nerve fiber of some; These Remak's nerve fibers can not be known in the effect of Roots of trigeminal nerve; But have circumstantial evidence to show, these Remak's nerve fibers have potential sensory function, and this maybe be relevant with radix sensoria nervi trigemini selective rhizotomy postoperative persistence of sensation and pain sensation alleviation failure.The area that Ezure etc. discover aixs cylinder in the Roots of trigeminal nerve and girth than sensory root greatly, the aixs cylinder distribution range is wider than sensory root, no thick a fiber in the sensory root.Sun Eryu etc. utilize adult's corpse head as sample, and the utilization surgery microscope has been done careful anatomical research to the trigeminal neuralgia intracranial segment, and motor root and sensation location of root relation have been discussed, and think to have ramus anastomoticus between motor root and the sensory root.But, do not see people embryo Roots of trigeminal nerve and motor root traveling correlative study report so far both at home and abroad as yet from the viewpoint of auxology.
Summary of the invention
To embryo's Roots of trigeminal nerve and the motor root traveling correlative study report of also having no talent at present.The present invention is based on people embryo trigeminal neuralgia and utilize tissue section strain to realize three-dimensional reconstruction, obtained effect preferably.
The present invention provides a kind of and utilizes tissue section strain to carry out three-dimensional rebuilding method based on people embryo trigeminal neuralgia.Through selecting people embryo fresh specimens, be the induced labor foetus of non-disease death especially, the gasserian ganglion of the gestational age people embryo fresh specimens that 20 weeks are above; Root and nervous ramification; Through the HE normal dyeing, utilize light border transmission electron microscope observing, adopt the computer realization three-dimensional reconstruction.
The present invention is through Roots of trigeminal nerve, joint and the HE of branch normal dyeing to sample; Utilize light microscopic, electron microscopic observation; Three-dimensional reconstruction, the research of method through the traveling of trifacial form of people embryo and motor root is carried out is for the first time the trifacial 3-D solid structure of people embryo to be shown on the human history; Both enriched the content of human trigeminal neuralgia morphology research, for clinical prosopalgic pathologic, physiologic and treatment morphologic basis was provided simultaneously.
The present invention provides specifically that a kind of to utilize tissue section strain to carry out the three-dimensional rebuilding method step based on people embryo trigeminal neuralgia following:
1. sample is fixed
After obtaining sample, in the 1-4h, with after the flushing of physiological saline heart, fixing through aortic perfusion with 10% formalin solution 1000-1500mL more earlier, keeps in Dark Place in dry place.
2. visual inspection
After treating that sample is completely fixed, get fetal head, open cranium, expose akrencephalon, win a side cerebral hemisphere and a diencephalon, the orthopaedic surgical operations microscopically removes the part midbrain, confirms that left side Roots of trigeminal nerve, joint and three big branches carry out big and small dissection and observation, carry out material object and take pictures.
3. electron microscopic observation
With the complete taking-up of the sample orthopaedic surgical operations microscopically right side Roots of trigeminal nerve, gasserian ganglion and the three big branches that obtain, behind the finishing tissue, naked eyes can be distinguished motor root of trigeminal nerve, peel off motor root of trigeminal nerve; Organize respectively with what get that mark places the container that 2.5% glutaraldehyde solution is housed well, the set time is 72 hours; Tissue that fixation mark is good takes out, and moves into the rinsing 3 times in PH7.4 of 1/15M phosphate buffer, each each 15 minutes; Starve in the sour immobile liquid back after fixing 1 hour, with being organized into 1% after the rinsing by as above getting into dehydration after the step rinsing; After entering 50% alcohol, 70% alcohol, 80% alcohol, 90% alcohol, 100% alcohol respectively dewatered 15 minutes, be placed in 100% alcohol and dewater; In v/v, adopt acetone and epoxy resin to soak in the liquid 1 hour according to 1: 1, acetone and epoxy resin in liquid soaked into 3 hours at 1: 3, soak into 12 hours in the pure epoxy resin after; Adopt the pure epoxy resin embedding, 37 ℃ 12 hours, 45 ℃ 12 hours, 60 ℃ 24 hours; Employing ultramicrotome section selects wherein that 1-2 opens section, under optical microscope, observes after the Toluidine blue staining, and according to the foundation that the Toluidine blue staining result provides, ultra-thin section is carried out at the more position of selection neuron again; Select for use the two colouring methods of uranium acetate and lead citrate that section is dyeed; Section is placed transmission electron microscope observing, gather picture.
4.HE dyeing
Each sample all with the filter paper parcel, with after soaking 12 hours in the crocus cloth parcel water, was gone into 75% dehydration of alcohol 4 hours outward; 85% dehydration of alcohol 4 hours; 90% alcohol spends the night, and is placed on 95% dehydration of alcohol 4 hours, and 100% dehydration of alcohol spends the night with 100% alcohol after 4 hours again; Went among the xylene I 30 minutes, and went among the xylene II till transparent; Go among the electric oven liquid wax I to pass through wax 1 hour, passed through wax 30 minutes among the liquid wax II; Take out sample and put into embedding frame wax oil and treat it and put into tap water after fixing and cool off, treat that wax stone is shaped after, wax stone is fixed mark, place refrigerator and cooled to hide, use during section; Select microtome for use, make serial section by coronal section, slice thickness is 6 μ m; The section of mounting is placed on the staining rack, put into 60 ℃ of baking boxs 30 minutes.Go in xylene I, II, the III liquid each after the taking-up 2 minutes, and went into 100% alcohol I, II, 95% alcohol, each 1-2 of 85% alcohol minute, use tap water, distilled water flushing a moment respectively.Su Dasu contaminated 5 minutes, and the tap water developing sheet is carved into 1% hydrochloride alcohol differentiation 3-5 second, and the tap water flushing for a moment.Ammoniacal liquor a moment, tap water flushing a moment to go into 0.5% Yihong solution and contaminated 5 minutes, 75% alcohol, 85% alcohol, 95% alcohol I are moments later gone in washing, II, 100% alcohol I, dehydration in each 1-2 of II minute reenters among xylene I, the II transparent each 1-2 minute.Neutral gum sealing section preparation is again with every biopsy marker ordering; Section is placed observation under the light microscopic, gather picture.
5. three-dimensional reconstruction
Utilize the HE coloration result, under light microscopic,, form the former figure of every section with the automated imaging technology in 40 times of visual field images acquired; Adopt similar Adobe photoshop instrument to cell, nerve root, each branch and the motor root etc. of each section with the various colors boundary line of drawing; Each that adopts that similar Adobe photoshop instrument finishes is opened section by from the bottom up rank order; With each section according to cell, three big branches, motion location of root to good; Draw a triangle on the tissue next door then; The position of this triangle from first to a last section must be consistent, and it is preserved with the psd form as a standard axle; With each image size 2446 * 2669 that the psd form is preserved, pixel 72,536KB changes 562 * 629 into, pixel 100,52.2KB, amended image is preserved with the jpg form.Every image that the jpg form is preserved is imported computing machine in order, select the slice thickness of rebuilding.
The used organization material of the present invention adopts the coronal section serial section, bed thickness 6 μ m since at interval three choose picture, so the bed thickness of actual reconstruction is 18 μ m, accomplishes the three-dimensional image reconstruction with TRI for Win 95/NT Version:3.10 software then.
Can clearly show the distribution of people embryo Roots of trigeminal nerve, joint and branch thereof through the three-dimensional image of the present invention's reconstruction; The piece cutting structure of people embryo Roots of trigeminal nerve, joint and the HE of branch dyeing is rebuild out, demonstrated the corresponding relation between the three better.From the picture of three-dimensional reconstruction, can find out people embryo's motor root of trigeminal nerve and sensory root companion row; Be positioned at the below of sensory root; And the dark face in 1/3 place is kept straight on below gradually in the trigeminal neuralgia semilunar ganglion, is parallel to the dark face of preceding inner edge of mandibular nerve afterwards, is slightly covered.The three big branches that the gasserian ganglion cell process is formed are respectively ophthalmic nerve, maxillary nerve and mandibular nerve under outside interior.
Through the concrete content of embodiment of the present invention, can reach following beneficial effect.
1, the three-dimensional image of the present invention's reconstruction can clearly show the distribution of people embryo Roots of trigeminal nerve, joint and branch thereof; 1/3 place under the concentrated inboard of motor root of trigeminal nerve, the neuromere through radix sensoria nervi trigemini; Be a continuous structure from the root to the mandibular nerve, incorporate the interior below of mandibular nerve at last into.Thereby show that the method that the present invention utilizes the sample of histotomy to carry out three-dimensional reconstruction is feasible.
2, traditional three-dimensional reconstruction has method not of the same race; The related personnel of non-computer specialty is difficult to the independent three-dimensional reconstruction of accomplishing; The inventive method is simple and practical, general laboratory technician, and amateur computernik can accomplish complicated histotomy three-dimensional reconstruction; And method is specifically feasible, can independently accomplish.
3, the present invention rebuilds through three-dimensional image; Can clearly see Roots of trigeminal nerve, joint and branch's 3-D solid structure especially can see also that from the dorsal part of sample the trigeminal neuralgia motor fiber is at Roots of trigeminal nerve; Position traveling within joint and the mandibular nerve; This provides the morphologic basis of aspects such as relevant dissection, histology to prosopalgic surgical intervention, and this operation is had certain theory and practice significance.
Description of drawings
Fig. 1 demonstration is the location drawing of people embryo Roots of trigeminal nerve, joint and three branches, and among the figure, V is a Roots of trigeminal nerve; V1 is an ophthalmic nerve; V2 is a maxillary nerve; V3 is a mandibular nerve; GN is a gasserian ganglion; II is an optic nerve; III is an oculomotor nerve; P is a pons; C is a cerebellum; M is a midbrain; T is an akrencephalon.
Fig. 2 is shown as Roots of trigeminal nerve, joint and the branch aspect graph under light microscopic, and under 4 * 10 times of conditions of HE dyeing, among the figure, Vr is a Roots of trigeminal nerve; V1 is an ophthalmic nerve; V2 is a maxillary nerve; V3 is a mandibular nerve; GN is a gasserian ganglion; Mr is the kinesitherapy nerve root.
Embodiment
Below will combine accompanying drawing that structure composition of the present invention and mode of operation thereof and principle are described further, still, the present invention is not limited to following embodiment.In addition, in following explanation, if no special instructions, % all refers to the V/V percent by volume.
Equipment and material have among the present invention:
Collect non-disease terminal pregnancy and the healthy fetus of induced labor, male or female, gestational age are more than 20 weeks, and gestational age combines the health check-up of sample to obtain according to pregnant woman's last menstrual period by ultrasound diagnosis.All samples of the present invention are fetched and delivered the laboratory at once all from the municipal district hospital, Urumchi after the fetus induced labor, earlier with behind the normal saline flushing, fix through aortic perfusion with 10% formalin solution 1000-1500mL again.Long for fear of the sample death time, tissue degeneratiaon influences test findings, and the present invention is controlled within 4 hours to time of perfusion fixation after with the sample induced labor.
Surgery microscope: No.1 Hospital Attached to Xinjiang Medical Univ. provides, micro-surgical instruments: Cao worker's board, the kind boat of Shandong Zibo engineering in medicine research institute makes, the routine operation apparatus: human dissection teaching and research room of Xinjiang Medicine University provides, fluorescent microscope: Xiamen Motic BA400 type, autopsy table: human dissection teaching and research room of Xinjiang Medicine University provides, digital camera: Japanese Nikon 4500 digital cameras (microspur 2cm), paraffin slicing machine: German Lycra 2135 types, transmission electron microscope: Japanese JEM-1230 type, ultramicrotome: Sweden LKB-2188 type, full microscope: Xiamen Motic BA600 type, refrigerator: Qingdao Haier, electronic analytical balance: autoclave is used in plum Teller-Tuo benefit Instr Ltd., MLS-3750 type laboratory: Sanyo Electric International Trading Company Ltd, three-dimensional reconstruction computing machine: the Japanese medical university second anatomy classroom
The reagent that relates among the present invention and the preparation of working fluid:
Chemical reagent: Dil:FMP FM003282, Japanese medical university provides, the PBS pulvis: Fuzhou Maixin biotechnology Development Co., Ltd, formalin analyze pure: Xi'an chemical reagent factory, absolute ethyl alcohol: Xi'an chemical reagent factory, xylene, Yihong, haematoxylin, sodium chloride, potassium chloride, sodium hydrogen phosphate, potassium dihydrogen phosphate etc. are pure for homemade analysis, distilled water, 2.5% glutaraldehyde, 10% formalin solution etc. provide for human dissection teaching and research room of Xinjiang Medicine University and Electron Microscopy Room.
All reagent selected for use among the present invention and instrument all are well known selecting for use, but do not limit enforcement of the present invention, and other reagent more well known in the art and equipment are all applicable to the enforcement of the following embodiment of the present invention.
Embodiment one: utilize tissue section strain to carry out three-dimensional rebuilding method based on people embryo trigeminal neuralgia
1. sample is fixed
After obtaining sample, in the 1-4h, clamp fetal cord, open the front wall with pliers; Expose pericardium, cut pericardium, fully expose heart; No. 7 infusion niidls are inserted root of ascending aorta, cut a little otch in right atrium, inject continuously after the capable heart of 0.9% physiological saline 500ml washes with the 50ml syringe with scissors; Inject 10% formalin solution 1500ml again, let immobile liquid, observe the fixed effect of sample along the capable perfusion fixation of body circulation approach; Seeing has immobile liquid to flow out sample abdominal distention, this good fixing effect of limbs hardening indicating from the mouth of sample, nose etc.; The sample that perfusion is good continues to be soaked in that to carry out back in 10% formalin solution of fresh configuration fixing, and dry locating kept in Dark Place.
2. visual inspection
After treating that 4 routine samples are completely fixed, get fetal head, open cranium; Expose akrencephalon, win a side cerebral hemisphere and a diencephalon, the orthopaedic surgical operations microscopically removes the part midbrain; Confirm that left side Roots of trigeminal nerve, joint and three big branches carry out big and small dissection and observation, carry out material object and take pictures, referring to accompanying drawing 1,2.
3. electron microscopic observation
(1) draws materials: the complete taking-up of above-mentioned 4 routine sample orthopaedic surgical operations microscopicallies right side Roots of trigeminal nerve, gasserian ganglion and three big branches; Behind the finishing tissue; Naked eyes can be distinguished motor root of trigeminal nerve, peel off motor root of trigeminal nerve, and traveling is in the kinesitherapy nerve root of trigeminal neuralgia sections.
(2) preceding fixing: that will get organizes respectively that mark places the container that 2.5% glutaraldehyde solution is housed well, and the set time is 72 hours.
(3) rinsing: tissue that fixation mark is good takes out, and moves into the rinsing 3 times in PH7.4 of 1/15M phosphate buffer, each each 15 minutes.
(4) back is fixing: after being organized in the 1% hungry sour immobile liquid after the rinsing, fix 1 hour.
(5) rinsing: operation steps is with (3).
(6) dehydration: go into 50% alcohol, 70% alcohol, 80% alcohol, 90% alcohol, 100% alcohol I and respectively dewatered 15 minutes, be placed among the 100% alcohol II.
(7) soak into: according to volume ratio, acetone and epoxy resin in liquid soaked into 1 hour at 1: 1, and acetone and epoxy resin in liquid soaked into 3 hours at 1: 3, soaked in the pure epoxy resin 12 hours.
(8) embedding: the pure epoxy resin embedding, 37 ℃ 12 hours, 45 ℃ 12 hours, 60 ℃ 24 hours.
(9) section: with Sweden LKB-2188 type ultramicrotome section, select wherein that 1-2 opens section, observation under optical microscope after the Toluidine blue staining, according to the foundation that the Toluidine blue staining result provides, ultra-thin section is carried out at the more position of selection neuron again.
(10) dyeing: select for use the two colouring methods of uranium acetate and lead citrate that section is dyeed.
(11) observe: section is placed Japanese JEM-1230 type transmission electron microscope observing, gather picture.
4.HE dyeing three-dimensional reconstruction
(1) draw materials: 4 routine samples, after waiting to be completely fixed, get fetal head; After the PBS rinsing is clean, remove scalp, take off braincap along zygomatic arch upper limb 1cm plane annular; Cut off the endocranium of braincap portion again along same plane, the flat cross-section brain stem of superior colliculus upper limb, excision bilateral akrencephalon and diencephalon; The careful endocranium that cuts off tentorium cerebelli and gasserian ganglion top; Will be together with the basis cranii of brain stem, Roots of trigeminal nerve, joint, the position have no change, place under the surgery microscope; 2 routine samples are confirmed the position of left side Roots of trigeminal nerve, gasserian ganglion and three big branch ophthalmic nerves, maxillary nerve, mandibular nerve; Other 2 routine samples are confirmed the position of right side Roots of trigeminal nerve, gasserian ganglion and three big branch ophthalmic nerves, maxillary nerve, mandibular nerve, cut off ophthalmic nerve, circular hole place cut-out maxillary nerve, oval foramen place cut-out mandibular nerve, basilar part of pons cut-out Roots of trigeminal nerve respectively at the superior orbital fissure place, careful complete Roots of trigeminal nerve, gasserian ganglion and the three big branches of peeling off; Behind the finishing tissue, be positioned in 10% formalin solution of fresh configuration.
(2) embedding: each sample with after soaking 12 hours in the crocus cloth parcel water, was gone into 75% dehydration of alcohol 4 hours outward all with the filter paper parcel; 85% dehydration of alcohol 4 hours, 90% alcohol spends the night, and is placed on 95% dehydration of alcohol 4 hours; 100% alcohol I dehydration 4 hours, 100% alcohol II spends the night; Went among the xylene I 30 minutes, and went among the xylene II till transparent; Go among the electric oven liquid wax I to pass through wax 1 hour, passed through wax 30 minutes among the liquid wax II; Take out sample and put into embedding frame wax oil and treat it and put into tap water after fixing and cool off, treat that wax stone is shaped after, wax stone is fixed mark, place refrigerator and cooled to hide, use during section.
(3) section: with section center Germany Lycra 2135 type microtomes, make serial section by coronal section, slice thickness is 6 μ m.Every routine Roots of trigeminal nerve, neuromere sample all have the serial section about 130 in the 4 routine samples.
(4) dyeing: the section that will mount places on the staining rack, puts into 60 ℃ of baking boxs 30 minutes.Go in xylene I, II, the III liquid each after the taking-up 2 minutes, and went into 100% alcohol I, II, 95% alcohol, each 1-2 of 85% alcohol minute, use tap water, distilled water flushing a moment respectively.Haematoxylin was contaminated 5 minutes, and the tap water developing sheet is carved into 1% hydrochloride alcohol differentiation 3-5 second, and the tap water flushing for a moment.Ammoniacal liquor a moment, tap water flushing a moment to go into 0.5% Yihong solution and contaminated 5 minutes, 75% alcohol, 85% alcohol, 95% alcohol I are moments later gone in washing, II, 100% alcohol I, dehydration in each 1-2 of II minute reenters among xylene I, the II transparent each 1-2 minute.Neutral gum sealing section preparation is again with every biopsy marker ordering.
(5) observe: section is placed under the light microscopic observe, gather picture.
5. three-dimensional reconstruction
(1) HE coloration result in 40 times of visual field images acquired, is formed the former figure of every section with automated imaging technology, totally 136 under light microscopic.
(2) with Adobe photoshop 7.0 to cell, nerve root, each branch and the motor root etc. of each section with the various colors boundary line of drawing.
(3) each that finish with Adobe photoshop 7.0 is opened section by from the bottom up rank order; With the position of each section to good; Like cell, three big branches, motor root etc., draw a triangle on the tissue next door then, the position of this triangle from first to a last section must be consistent; It is preserved with the psd form as a standard axle.
(4) each pictures life size 2446 * 2669, pixel 72,536KB changes 562 * 629 into, pixel 100,52.2KB must preserve with the jpg form.
(5) accomplish three-dimensional image with TRI for Win 95/NT Version:3.10.
Embodiment two: sample is fixed
After obtaining sample, in the 1-4h, clamp fetal cord, open the front wall with pliers; Expose pericardium, cut pericardium, fully expose heart; No. 7 infusion niidls are inserted root of ascending aorta, cut a little otch in right atrium, inject continuously after the capable heart of 0.9% physiological saline 500ml washes with the 50ml syringe with scissors; Inject 10% formalin solution 1500ml again, let immobile liquid, observe the fixed effect of sample along the capable perfusion fixation of body circulation approach; Seeing has immobile liquid to flow out sample abdominal distention, this good fixing effect of limbs hardening indicating from the mouth of sample, nose etc.; The sample that perfusion is good continues to be soaked in that to carry out back in 10% formalin solution of fresh configuration fixing, and dry locating kept in Dark Place.
Embodiment three: light microscopic, transmission electron microscope observing
(1) draws materials: the complete taking-up of the foregoing description sample orthopaedic surgical operations microscopically right side Roots of trigeminal nerve, gasserian ganglion and three big branches; Behind the finishing tissue; Naked eyes can be distinguished motor root of trigeminal nerve; Peel off motor root of trigeminal nerve, traveling is in the kinesitherapy nerve root of trigeminal neuralgia sections, referring to accompanying drawing 1,2.
(2) preceding fixing: that will get organizes respectively that mark places the container that 2.5% glutaraldehyde solution is housed well, and the set time is 72 hours.
(3) rinsing: tissue that fixation mark is good takes out, and moves into the rinsing 3 times in PH7.4 of 1/15M phosphate buffer, each each 15 minutes.
(4) back is fixing: after being organized in the 1% hungry sour immobile liquid after the rinsing, fix 1 hour.
(5) rinsing: operation steps is with (3).
(6) dehydration: go into 50% alcohol, 70% alcohol, 80% alcohol, 90% alcohol, 100% alcohol I and respectively dewatered 15 minutes, be placed among the 100% alcohol II.
(7) soak into: according to volume ratio, acetone and epoxy resin in liquid soaked into 1 hour at 1: 1, and acetone and epoxy resin in liquid soaked into 3 hours at 1: 3, soaked in the pure epoxy resin 12 hours.
(8) embedding: the pure epoxy resin embedding, 37 ℃ 12 hours, 45 ℃ 12 hours, 60 ℃ 24 hours.
(9) section: with Sweden LKB-2188 type ultramicrotome section, select wherein that 1-2 opens section, observation under optical microscope after the Toluidine blue staining, according to the foundation that the Toluidine blue staining result provides, ultra-thin section is carried out at the more position of selection neuron again.
(10) dyeing: select for use the two colouring methods of uranium acetate and lead citrate that section is dyeed.
(11) observe: section is placed Japanese JEM-1230 type transmission electron microscope observing, gather picture.
Embodiment four: HE dyeing
(1) draw materials: 4 routine samples, after waiting to be completely fixed, get fetal head; After the PBS rinsing is clean, remove scalp, take off braincap along zygomatic arch upper limb 1cm plane annular; Cut off the endocranium of braincap portion again along same plane, the flat cross-section brain stem of superior colliculus upper limb, excision bilateral akrencephalon and diencephalon; The careful endocranium that cuts off tentorium cerebelli and gasserian ganglion top; Will be together with the basis cranii of brain stem, Roots of trigeminal nerve, joint, the position have no change, place under the surgery microscope; 2 routine samples are confirmed the position of left side Roots of trigeminal nerve, gasserian ganglion and three big branch ophthalmic nerves, maxillary nerve, mandibular nerve; Other 2 routine samples are confirmed the position of right side Roots of trigeminal nerve, gasserian ganglion and three big branch ophthalmic nerves, maxillary nerve, mandibular nerve, cut off ophthalmic nerve, circular hole place cut-out maxillary nerve, oval foramen place cut-out mandibular nerve, basilar part of pons cut-out Roots of trigeminal nerve respectively at the superior orbital fissure place, careful complete Roots of trigeminal nerve, gasserian ganglion and the three big branches of peeling off; Behind the finishing tissue, be positioned in 10% formalin solution of fresh configuration.
(2) embedding: each sample with after soaking 12 hours in the crocus cloth parcel water, was gone into 75% dehydration of alcohol 4 hours outward all with the filter paper parcel; 85% dehydration of alcohol 4 hours, 90% alcohol spends the night, and is placed on 95% dehydration of alcohol 4 hours; 100% alcohol I dehydration 4 hours, 100% alcohol II spends the night; Went among the xylene I 30 minutes, and went among the xylene II till transparent; Go among the electric oven liquid wax I to pass through wax 1 hour, passed through wax 30 minutes among the liquid wax II; Take out sample and put into embedding frame wax oil and treat it and put into tap water after fixing and cool off, treat that wax stone is shaped after, wax stone is fixed mark, place refrigerator and cooled to hide, use during section.
(3) section: with section center Germany Lycra 2135 type microtomes, make serial section by coronal section, slice thickness is 6 μ m.Every routine Roots of trigeminal nerve, neuromere sample all have the serial section about 130 in the 4 routine samples.
(4) dyeing: the section that will mount places on the staining rack, puts into 60 ℃ of baking boxs 30 minutes.Go in xylene I, II, the III liquid each after the taking-up 2 minutes, went into 100% alcohol I, 100% alcohol II, 95% alcohol, each 1-2 of 85% alcohol minute, use tap water, distilled water flushing a moment respectively.Haematoxylin was contaminated 5 minutes, and the tap water developing sheet is carved into 1% hydrochloride alcohol differentiation 3-5 second, and the tap water flushing for a moment.Ammoniacal liquor a moment, tap water flushing a moment, go into 0.5% Yihong solution and contaminated 5 minutes; 75% alcohol, 85% alcohol, 95% alcohol I are moments later gone in washing; 95% alcohol II, 100% alcohol I, dehydration in each 1-2 of II minute reenters among xylene I, the II transparent each 1-2 minute.Neutral gum sealing section preparation is again with every biopsy marker ordering.
(5) observe: section is placed under the light microscopic observe, gather picture.
Embodiment five: three-dimensional reconstruction
(1) HE coloration result in 40 times of visual field images acquired, is formed the former figure of every section with automated imaging technology, totally 136 under light microscopic.
(2) with Adobe photoshop 7.0 to cell, nerve root, each branch and the motor root etc. of each section with boundary line, various colors picture place.
(3) each that finish with Adobe photoshop 7.0 is opened section by from the bottom up rank order; With the position of each section to good; Like cell, three big branches, motor root etc., draw a triangle on the tissue next door then, the position of this triangle from first to a last section must be consistent; It is preserved with the psd form as a standard axle.
(4) each pictures life size 2446 * 2669, pixel 72,536KB changes 562 * 629 into, pixel 100,52.2KB must preserve with the jpg form.
(5) accomplish three-dimensional image with TRI for Win 95/NT Version:3.10.
Claims (2)
1. one kind is utilized tissue section strain to carry out three-dimensional rebuilding method based on people embryo trigeminal neuralgia; Through Roots of trigeminal nerve, joint and branch's dyeing to sample; Utilize light microscopic, electron microscopic observation, three-dimensional reconstruction is characterized in that; According to the percent by volume meter, described to utilize tissue section strain to carry out the three-dimensional reconstruction concrete steps based on people embryo trigeminal neuralgia following:
(1) sample is fixed: after obtaining sample, in the 1-4h, with after the flushing of physiological saline heart, fixing through aortic perfusion with 10% formalin solution 1000-1500mL more earlier, keeps in Dark Place in dry place;
(2) visual inspection: after treating that sample is completely fixed, get fetal head, open cranium; Expose akrencephalon, win a side cerebral hemisphere and a diencephalon, the orthopaedic surgical operations microscopically removes the part midbrain; Confirm that left side Roots of trigeminal nerve, joint and three big branches carry out big and small dissection and observation, carry out material object and take pictures;
(3) electron microscopic observation: with the complete taking-up of the sample orthopaedic surgical operations microscopically right side Roots of trigeminal nerve, gasserian ganglion and the three big branches that obtain, behind the finishing tissue, naked eyes can be distinguished motor root of trigeminal nerve, peel off motor root of trigeminal nerve; Organize respectively with what get that mark places the container that 2.5% glutaraldehyde solution is housed well, the set time is 72 hours; Tissue that fixation mark is good takes out, and moves into the rinsing 3 times in PH7.4 of 1/15M phosphate buffer, each each 15 minutes; Starve in the sour immobile liquid back after fixing 1 hour, with being organized into 1% after the rinsing by as above getting into dehydration after the step rinsing; After entering 50% alcohol, 70% alcohol, 80% alcohol, 90% alcohol, 100% alcohol respectively dewatered 15 minutes, place 100% alcohol to dewater; In v/v, adopt acetone and epoxy resin to soak in the liquid 1 hour according to 1: 1, acetone and epoxy resin in liquid soaked into 3 hours at 1: 3, soak into 12 hours in the pure epoxy resin after; Adopt the pure epoxy resin embedding, 37 ℃ 12 hours, 45 ℃ 12 hours, 60 ℃ 24 hours; Employing ultramicrotome section selects wherein that 1-2 opens section, under optical microscope, observes after the Toluidine blue staining, and according to the foundation that the Toluidine blue staining result provides, ultra-thin section is carried out at the more position of selection neuron again; Select for use the two colouring methods of uranium acetate and lead citrate that section is dyeed; Section is placed transmission electron microscope observing, gather picture;
(4) HE dyeing: each sample all with the filter paper parcel, with after soaking 12 hours in the crocus cloth parcel water, was gone into 75% dehydration of alcohol 4 hours outward; 85% dehydration of alcohol 4 hours; 90% alcohol spends the night, and is placed on 95% dehydration of alcohol 4 hours, and 100% dehydration of alcohol spends the night with 100% alcohol after 4 hours again; Went among the xylene I 30 minutes, and went in the xylene II till transparent; Go in the electric oven liquid wax I to pass through wax 1 hour, passed through wax 30 minutes in the liquid wax II; Take out sample and put into embedding frame wax oil and treat it and put into tap water after fixing and cool off, treat that wax stone is shaped after, wax stone is fixed mark, place refrigerator and cooled to hide, use during section; Select microtome for use, make serial section by coronal section, slice thickness is 6 μ m; The section of mounting is placed on the staining rack, put into 60 ℃ of baking boxs 30 minutes; Go in xylene I, II, the III liquid each after the taking-up 2 minutes, and went into 100% alcohol I, alcohol II, 95% alcohol, each 1-2 of 85% alcohol minute, use tap water, distilled water flushing a moment respectively; Haematoxylin was contaminated 5 minutes, and the tap water developing sheet is carved into 1% hydrochloride alcohol differentiation 3-5 second, and the tap water flushing for a moment; Ammoniacal liquor for a moment; The tap water flushing for a moment; Go into 0.5% Yihong solution and contaminated 5 minutes, 75% alcohol, 85% alcohol, 95% alcohol I, 95% alcohol II, 100% alcohol I, 100% each 1-2 of alcohol II minute dehydration are moments later gone in washing, reenter in xylene I, the II transparent each 1-2 minute; Neutral gum sealing section preparation is again with every biopsy marker ordering; Section is placed observation under the light microscopic, gather picture;
(5) three-dimensional reconstruction: utilize the HE coloration result, under light microscopic,, form the whole former figure of every section with the automated imaging technology in 40 times of visual field images acquired; Adopt Adobe photoshop instrument to cell, nerve root, each branch and the motor root of each section with the various colors boundary line of drawing; Each that adopts that Adobe photoshop instrument finishes is opened section by from the bottom up rank order; With each section according to cell, three big branches, motion location of root to good; Draw a triangle on the tissue next door then; The position of this triangle from first to a last section must be consistent, and it is preserved with the psd form as a standard axle; With each image size 2446 * 2669 that the psd form is preserved, pixel 72,536KB changes 562 * 629 into, pixel 100,52.2KB, amended image is preserved with the jpg form; Every image that the jpg form is preserved is imported computing machine in order, select the slice thickness of rebuilding.
2. utilize tissue section strain to carry out three-dimensional rebuilding method like claim 1 based on people embryo trigeminal neuralgia; It is characterized in that; Describedly utilize histotomy to adopt the coronal section serial section based on people embryo trigeminal neuralgia, bed thickness 6 μ m choose picture for three at interval; The bed thickness of rebuilding is 18 μ m, accomplishes three-dimensional image with TRI for Win 95/NT Version:3.10 software then and rebuilds.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018205691A1 (en) * | 2017-05-09 | 2018-11-15 | 南昌德漫多科技有限公司 | System for processing histopathological specimen |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9008378B2 (en) * | 2006-12-20 | 2015-04-14 | The Board Of Trustees Of The Leland Stanford Junior University | Arrangement and imaging of biological samples |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1846614A (en) * | 2005-04-04 | 2006-10-18 | 沈渊瑶 | Method of constructing 3D tissue image |
| CN101246159A (en) * | 2008-03-07 | 2008-08-20 | 华中科技大学 | Method for fast identifying peripheral nerve bundle nature |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005007233A2 (en) * | 2003-06-20 | 2005-01-27 | Massachusetts Institute Of Technology | Application of electrical stimulation for functional tissue engineering in vitro and in vivo |
| US8649576B2 (en) * | 2006-06-16 | 2014-02-11 | George Mason Intellectual Properties, Inc. | Arborization reconstruction |
-
2010
- 2010-07-07 CN CN2010102191679A patent/CN101915693B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1846614A (en) * | 2005-04-04 | 2006-10-18 | 沈渊瑶 | Method of constructing 3D tissue image |
| CN101246159A (en) * | 2008-03-07 | 2008-08-20 | 华中科技大学 | Method for fast identifying peripheral nerve bundle nature |
Non-Patent Citations (4)
| Title |
|---|
| Alexandra Borges et al..Imaging the trigeminal nerve.《European Journal of Radiology》.2010,第74卷(第2期),323-340. * |
| Tatsurou Tanaka et al..Utility of magnetic resonance cisternography using three-dimensional fast asymmetric spin-echo sequences with multiplanar reconstruction: The evaluation of sites of neurovascular compression of the trigeminal nerve.《Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology, and Endodontology》.2005,第100卷(第2期),215–225. * |
| 吐尔逊江·达地汗等.人胚胎三叉神经运动纤维的定位研究.《新疆医科大学学报》.2009,第32卷(第8期),1043-1044，1048. * |
| 吴励等.人胚胎三叉神经节细胞电镜及光镜观察.《现代生物医学进展》.2009,第9卷(第5期),881-883. * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018205691A1 (en) * | 2017-05-09 | 2018-11-15 | 南昌德漫多科技有限公司 | System for processing histopathological specimen |
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