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貨號	品名	包裝	品牌
78000	熒光金 Fluoro-Gold	20mg	fluorochrome

熒光金逆行標(biāo)記大鼠視網(wǎng)膜神經(jīng)節(jié)細(xì)胞

【摘要】目的將熒光金（flurogold,FG）注射于SD大鼠上丘，觀察到被FG逆行標(biāo)記的視網(wǎng)膜神經(jīng)節(jié)細(xì)胞(retinal ganglion cells,RGC)，以及SD大鼠RGC的數(shù)目及分布。方法用3%FG雙上丘注射，逆行標(biāo)記RGC。5天后做視網(wǎng)膜定向鋪片，在熒光顯微鏡下距離視乳頭中心上下左右各2mm拍攝照片。利用計算機(jī)圖像分析系統(tǒng)做RGC計數(shù)。結(jié)果右眼RGC單位面積數(shù)量472.9±54.13，左眼RGC單位面積數(shù)量471.5±52.54。雙眼RGC單位面積數(shù)量相比差異無顯著性（P>0.05）。視網(wǎng)膜血管自視盤發(fā)出呈放射狀，血管走行區(qū)無標(biāo)記細(xì)胞。距視盤2mm始RGC分布較致密，視網(wǎng)膜周邊部細(xì)胞分布較為稀疏。結(jié)論用熒光金逆行標(biāo)記上丘方法來標(biāo)記RGC，是實驗條件下研究RGC數(shù)量動態(tài)變化的可靠、有效方法。

　　【關(guān)鍵詞】視網(wǎng)膜神經(jīng)節(jié)細(xì)胞;熒光金

　　Retinal ganglion cells retrograde labelled by injection of fluorogold

　　LI Yue-hua，MA Ke，Xu Liang.

　　Department of Ophthalmology,Beijing Chaoyang Hospital,Beijing 100020，China

　　【Abstract】 Objective Retinal ganglion cells (RGC) were retrograde labeled by injection of 3% fluorogold into both side of superior colliculus to observe the numbers and distribution of RGC .Four photographs were taken from every quadrant of the retina 2mm from the center of optic disc. Retinal ganglion cells were quantitatively analyzed by computer.Methods To identify RGC,we applied the 3%fluorogold to the superior colliculi.The retinal were examined through a fluorescence microscope after 5 days,four photographs were taken from every quadrant of the retina 2mm from the center of optic disc. Retinal ganglion cells were quantitatively analyzed by computer.Results The numbers of RGC were not significantly different between two groups. The numbers of right eye was 472.9±54.13.The numbers of left eye was 471.5±52.54.Conclusion The method that retinal ganglion cells (RGC) retrograde labeled by injection of fluorogold is reliable and effective.

　　【Key words】 retinal ganglion cells;fluorogold

視網(wǎng)膜神經(jīng)節(jié)細(xì)胞（retinal ganglion cells,RGC）的慢性進(jìn)行性丟失是青光眼zui主要的病理生理學(xué)特征。高眼壓、缺血等原發(fā)性損傷首先引起一部分RGC的丟失，丟失的節(jié)細(xì)胞產(chǎn)生出許多毒性物質(zhì)，使其周圍RGC的生存環(huán)境惡化，細(xì)胞膜離子通透性增加，線粒體膜遭受損害，致使細(xì)胞進(jìn)入凋亡程序，導(dǎo)致細(xì)胞丟失［1］。因此準(zhǔn)確的RGC計數(shù)是研究青光眼發(fā)病機(jī)制及視神經(jīng)保護(hù)治療的關(guān)鍵指標(biāo)。該實驗將熒光金（flurogold,FG）注射于SD大鼠上丘，觀察到被FG逆行標(biāo)記的RGC，以及SD大鼠RGC的數(shù)目及分布。

　　1 材料與方法

　　1.1 試劑與器械熒光金（熒光金公司，美國），腦立體定位儀900型（David Kopf儀器公司，美國），熒光顯微鏡（AH-2，奧林巴斯公司，日本），聯(lián)想Mustek掃描儀。

　　1.2 實驗動物 Sprague-Dawley大鼠10只，購自北京維通利華實驗動物技術(shù)有限公司。雄性，重量200～240g,裂隙燈檢查大鼠前節(jié)，包括角膜、前房、虹膜和晶狀體無異常改變。Mydrine散瞳，檢眼鏡觀察眼底，包括視盤、眼底血管及部分視網(wǎng)膜無異常改變者。實驗動物在12h光照/12h黑暗的條件下飼養(yǎng)。

　　1.3 逆行標(biāo)記采用雙上丘注射法逆行標(biāo)記RGC。大鼠腹腔注射進(jìn)行麻醉，將其固定在腦立體定位儀上，0.5%碘伏消毒皮膚，于正中切開皮膚向下分離達(dá)顱骨。牙科鉆鉆開顱骨相應(yīng)部位，用微量注射器吸取3%FG（溶于0.9%生理鹽水中），每側(cè)上丘注射兩點（前囟后5.9 和 6.4mm，旁開1.4mm，深4.0mm），每點注射1.5μl，留針5min。

　　1.4 視網(wǎng)膜定向鋪片實驗第5天，實驗動物在深麻醉下，于上方12點處做球結(jié)膜縫線標(biāo)記，立即取出眼球固定于4%多聚甲醛磷酸緩沖液中2h。沿角膜緣后0.5mm剪開眼球，去除角膜和晶狀體，分離視網(wǎng)膜并將其定向平鋪，空氣中自然干燥，市售無色指甲油封片。

　　1.5 熒光照片RGC計數(shù) 在熒光顯微鏡下使用V波段熒光激發(fā)，距視盤中心2mm上下左右各拍攝照片1張，照片放大倍數(shù)125×，分別代表上、下、左、右四個象限的RGC數(shù)量。

　　1.6 圖像分析使用聯(lián)想Mustek掃描儀將照片以100dpi掃入計算機(jī)，使用彩色顆粒分析軟件（CPAS）進(jìn)行節(jié)細(xì)胞計數(shù)，將上、下、左、右4張照片節(jié)細(xì)胞數(shù)量累加，代表該眼節(jié)細(xì)胞單位面積數(shù)量。

　　1.7 統(tǒng)計學(xué)方法使用SPSS for Windows 10.0統(tǒng)計軟件包，采用配對t檢驗，P<0.05差異具有顯著性，P<0.01差異具有非常顯著性。

　　2 結(jié)果

　　2.1 FG標(biāo)記的RGC的形態(tài) 在FG標(biāo)記后5天，大鼠視網(wǎng)膜鋪片觀察到標(biāo)記的RGC細(xì)胞質(zhì)中熒光均勻，邊界清晰，有些細(xì)胞可見有熒光充盈的細(xì)胞突起，細(xì)胞外無熒光染料滲漏（見圖1）。
　　2.2 RGC計數(shù)及分布雙眼RGC單位面積數(shù)量見表1。兩眼相比較差異無顯著性（P>0.05）。視網(wǎng)膜血管自視盤發(fā)出呈放射狀，血管走行區(qū)無標(biāo)記細(xì)胞。距視盤2mm始RGC分布較致密，視網(wǎng)膜周邊部細(xì)胞分布較為稀疏（見圖2）。但細(xì)胞內(nèi)熒光仍較明顯。
　　表1 雙眼RGC單位面積數(shù)量　略
　　3 討論

　　利用軸漿流逆向輸送的特點，在顱內(nèi)RGC投射的核團(tuán)上丘、外側(cè)膝狀體［2］、視神經(jīng)斷端近眼球一側(cè)放置辣根過氧化物酶或Diamidido Yellow，F(xiàn)G(Fluorogold),Evan Blue,Fluoro-Ruby(FR)［ 2～5］等各種熒光物質(zhì)，通過逆向軸漿流將這些物質(zhì)帶到RGC胞體內(nèi)，產(chǎn)生特定的染色效果，標(biāo)記出RGC，而RGC層中的其他細(xì)胞則不能著色。而HRP參與細(xì)胞代謝，不能在細(xì)胞內(nèi)長期存留。某些示蹤劑如快藍(lán)、核黃等，易于從標(biāo)記細(xì)胞內(nèi)擴(kuò)散到周圍組織，且照射時褪色較快，即使保存在低溫、避光條件下，仍不能長期保存。另外應(yīng)用傳統(tǒng)的組織化學(xué)技術(shù)無法將RGC與視網(wǎng)膜內(nèi)其他神經(jīng)元嚴(yán)格區(qū)分，尤其是與移位的無長突細(xì)胞鑒別。利用軸漿流逆向輸送的特點，目前多用熒光金逆行標(biāo)記上丘方法來標(biāo)記RGC，是實驗條件下研究RGC數(shù)量動態(tài)變化的可靠、有效方法。
　　熒光金在紫外線照射下，其激發(fā)光波長323nm,發(fā)射光波長為408nm，標(biāo)記的RGC呈金黃色強(qiáng)熒光，能標(biāo)記細(xì)胞質(zhì)，而細(xì)胞核不著色，能很好顯示樹突分支，細(xì)胞外無熒光染料滲漏，不易擴(kuò)散，與周圍組織分界清晰，除RGC層外，其余各層中均無熒光標(biāo)記的細(xì)胞。Selles-Navarro I［6］等研究發(fā)現(xiàn)應(yīng)用熒光金行上丘逆行標(biāo)記RGC后，細(xì)胞質(zhì)內(nèi)熒光金的存在不超過3周，標(biāo)記后12天、14天、21天與標(biāo)記后37天有明顯差異。隨時間進(jìn)展熒光金可能丟失熒光或被代謝［7］。該實驗于動物處死前5天行熒光金逆行標(biāo)記上丘，標(biāo)記的RGC呈金黃色強(qiáng)熒光，各象限均勻。通過逆行標(biāo)記RGC可以記錄它的累積丟失，比通過凋亡標(biāo)記（如TUNEL染色）更好，通過凋亡標(biāo)記在一定的時間只能看到少量細(xì)胞［8］。此標(biāo)記方法廣泛應(yīng)用于RGC的發(fā)生和凋亡［9］、視網(wǎng)膜缺血［6］、視神經(jīng)橫斷、視神經(jīng)再生的研究。
該實驗的標(biāo)記方法為兩點法雙側(cè)上丘注射，因為有95%以上的大鼠RGC投射到上丘［10］，10%左右的RGC的軸突投射到同側(cè)外側(cè)膝狀體。因此，采用對側(cè)上丘注射標(biāo)記單眼RGC的方法并不可靠，至少有10%的RGC不被標(biāo)記。上丘注射法還包括一點法［10］、三點法［11］。以往我們也采取過每側(cè)上丘注射一點的標(biāo)記方法，發(fā)現(xiàn)這種方法能夠成功標(biāo)記RGC的比率比較低，而且對注射精度要求高，注射點必須位于上丘中心（前囟后5.3mm中線外2mm,深4.5mm）。如果注射偏離上丘中心位置，將導(dǎo)致視網(wǎng)膜RGC標(biāo)記的不均勻。改用雙側(cè)上丘兩點注射法標(biāo)記出的RGC分布均勻，對視網(wǎng)膜各象限RGC數(shù)的統(tǒng)計學(xué)檢驗差異無顯著性。另外發(fā)現(xiàn)RGC數(shù)量從中心到視網(wǎng)膜周邊并沒有大幅度衰減。三點標(biāo)記法耗時長、增加動物感染機(jī)會、損傷較兩點法重。比較上丘一點注射法、兩點注射法和多點注射法，認(rèn)為兩點注射法效率高，標(biāo)記良好，有比較好的穩(wěn)定性。
實驗證明用熒光金逆行標(biāo)記上丘方法來標(biāo)記RGC，是實驗條件下研究RGC數(shù)量動態(tài)變化的可靠、有效方法?！　?/span>
【參考文獻(xiàn)】
1 Schwartz M, Belkin M, Yoles E, et al. Potential treatment modalities for glaucomatous neuropathy: neuroprotection and neuroregeneration . J Glaucoma, 1996，5: 427-432.
2 Kondo Y, Takada M, Honda Y, et al .Bilateral projections of single retinal ganglion cells to the lateral geniculate nuclei and superior colliculi in the albino rat. Brain Res, 1993，608(2): 204-215.
3 Farid Ahmed AK, Dong K, Setsu T, et al. Correlation between different types of retinal ganglion cells and their projection pattern in the albino rat. Brain Res, 1996，706(1): 163-168.
4 Farid Ahmed AK,Dong K, Hanna-Georges FB, et al. Retrograde double-labeling study of retinal ganglion cells from the ipsilateral VLGN and SC in the albino rat. Neurosci Lett, 1998，244(1): 47-51.
5 Levkovitch-Verbin H, Harris-Cerruti C,Groner Y, et al. RGC death in mice after optic nerne crush injury: oxidative stress and neuroprotection. Invest Ophthalmol Vis Sci, 2000，41(13): 4169-4174.
6 Selles-Navarro I, Villegas-Perez MP, Salvador-Silva M, et al. Retinal ganglion cell death after different transient periods of pressure-induced ischemia and survival intervals. A quantitative in vivo study. Invest Ophthalmol Vis Sci, 1996，37(10): 2002-2014.
7 G mez Ram rez AM,Villegas-p rez MP,Salvador M，et al.Use the flrorescent tracer fluorogold to identify the motoneuron population of the abducens nucleus:a quantitative in vivo study . Invest Ophthalmol Vis Sci, 1995， 36: 687.
8 Garcia-Valenzuela E,Shareef S,Walsh J，et al.Programmed cell death of retinal ganglion cells during experimental glaucoma.Exp Eye Res, 1995，61: 33-44.
9 Lagreze WA, Knorle R, Bach M, et al. Memantine is neuroprotective in a rat model of pressure-induced retinal ischemia. Invest Ophthalmol Vis Sci, 1998, 39 Devil : 1063-1066.
10 Levkovitch-Verbin H,Harris-Cerruti C,Groner Y,et al.RGC death in mice after optic nerve crush injury:oxidative stress and neuroprotection. Invest Ophthalmol Vis Sci, 2000,41(13): 4169-4174.
11 Yoles E,Muller S,Schwartz M. NMDA-Receptor antagonist protects neurons from secondary degeneration after partial optic nerve crush.J Neurotrauma, 1997,14(9): 665-675.

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熒光金說明書

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Fluoro-Gold Protocol and Use Guide

Main Protocol
1. Background
The use of Fluoro-Gold is essentially the same as other fluorescent tracers. The main difference is that Fluoro-Gold is more flexible in terms of post-injection survival times, concentration range, tissue treatment and compatibility with other histochemical techniques.

2. Storage and Shelf Life
Dry Fluoro-Gold should be kept in a light tight closed container at 4 degrees Celsius. Stored properly, Fluorogold should have a shelf life exceeding one year. The dye in solution should also be kept in a light tight closed container at 4 degrees Celsius and should remain stable for at least six months.

3. Vehicle
Fluoro-Gold can be dissolved in distilled water or 0.9% saline, or utilized as a suspension
in 0.2M neutral phosphate buffer.

4. Dye Concentration
Fluoro-Gold has been successfully used at concentrations ranging from 1-10%. Initially, a 4% concentration is advised. If undesirable necrosis occurs at the injection site, or labeling is too intense, reduce the concentration to a 2% solution. If you need to use more precise measurements, the molecular weight of Fluoro-Gold is 532.6 daltons.

5. Dye Administration

A. Pressure Injection - This is probably the most frequently used mode of application. Volumes injected range from .05-1 µ l, typically .1-.2 µ l.

B. Iontophoresis - Discrete, small injection sites result from 4-10 second pulsed iontophoretic (+5 to +10ua/10min) application.

C. Crystal - A crystal of the tracer can be administered from the tip of a micro-pipette.

6. Post-0perative Survival Period
Good retrograde labeling has been observed with periods ranging from two days to two months. Survival periods of three to five days are typical. Long survival periods enhance filling of distal processes without diffusion of the dye from the cell.

7. Fixation
Almost any fixative, or no fixative, can be used, Phosphate neutral buffered saline containing 4% formaldehyde is frequently employed. Fixatives containing high concentrations of heavy metals (e.g. osmium, mercury) will quench the fluorescence, while high concentrations (over 1%) of glutaraldehyde may increase background fluorescence

8. Histochemical Processing
Tissue containing Fluoro-Gold may be processed according to virtually any common histological technique. This includes cryostat sections of unfixed tissue (10 µm), frozen sections of fixed tissue (20 µm), and thin sections cut from tissue imbedded in either plastic (.2-4 µm) or paraffin (3-10 µm). Frozen sections of fixed tissue are most frequently used.

9. Combined Methods
At this point of processing, sections may be further processed for a second marker such as autoradiography, HRP histochemistry, immunocytochemistry, a second fluorescent tracer, fluorescent counterstain, etc.

10. Mounting, Clearing and Coverslipping
Sections are typically mounted on gelatin-coated slides, air-dried, immersed in xylene, and coverslipped with nonfluorescent DPX plastic mounting media. Sections may be dehydrated with graded alcohols, unless this is not compatible with a second tracer. If Fluoro-Gold is to be combined with fluorescence immunocytochemistry, then sections are air-dried and directly coverslipped with neutral buffered glycerine (1:2).

11. Examination and Photography
Fluoro-Gold can be visualized with a fluorescence microscope using a wide band ultraviolet excitation filter. A gold color is emitted when tissue has been processed with neutral pH buffer, whereas a blue color is emitted when tissue is processed with acidic (e.g. pH 3.3) pH buffer. It can be photographed digitally or with film (use Ektachrome 200-400 ASA film for color prints and comparable speed film for black and white prints, for example Tri-X). Most exposure times range from 10-60 second exposures, depending on the objective magnification and the intensity of the label. Thirty (30) second exposures are about average. Multiple exposures may be exploited to simultaneously visualize Fluoro-Gold and another tracer. Thus, UV would be combined with bright field illumination to simultaneously locate Fluoro-Gold with HRP or silver grains in autoradiography. Similarly, blue light excitation can be combined to also visualize the green emission color of FITC, while green excitation light may be used to simultaneously observe the red emission color of propidium iodide, or ethidium bromide (a fluorescent counterstain).

Additional Information Concerning the Use of Fluoro-Gold
Vehicle
For pressure injections through a microsyringe or micropipette, Fluoro-Gold should be dissolved in distilled water or .9% saline. Fluoro-Gold may also be utilized as a suspension in .2M neutral phosphate buffer, however, the suspended particles may clog a fine micropipette tip so distilled water or .9% saline is the preferred vehicle. For iontophoresis, a 1% Fluoro-Gold solution is made up in .1M acetate buffer (pH=3.3). Well-cleaned (95% ETOH, water) glass micropipettes should have tips of 10-20 µm. Optimal iontophoresis parameters are +1 to +5u amps delivered with pulsed current (4-10 seconds on, 4-10 seconds off) over a 10-20 minute period.

Injection Sites
Virtually any central or peripheral nervous system structure can be injected with Fluoro-Gold for analysis of retrograde transport. In the peripheral nervous system, ganglia and peripheral targets can be studied. For studies of peripheral nerve, the nerve should be cut or damaged and either dipped in, or injected with, aq 5% solution of Fluoro-Gold. Since Fluoro-Gold is not significantly taken up by intact fibers of passage, the fibers must be cut or severely damaged for uptake of the dye to occur.

Transport and Survival Time
Fluoro-Gold is used as a retrograde axonal tracer, although orthograde axonal transport does occur. The survival time should be varied (especially to very short survival times of 12 hours - 2 days) to maximize orthograde transport in the specific neuronal system under study. For retrograde transport, the survival times should be varied from 4 days to 14 days. Seven to 10 days works for most systems, although long pathways (e.g., spinal cord to brainstem) and pathways in large mammals (e.g., cats, monkeys) may require longer survival times (e.g., 14 days). In addition, since Fluoro-Gold remains fast within retrogradely labeled neurons, survival times of several months will also produce excellent results. For iontophoresis, a 2-5 day survival time is recommended. It is estimated that transport occurs at about 2 cm per day for mammals; it is slower for cold-blooded animals.

Tissue Processing
Tissue processing is covered in detail in the use guide and in the original publication (Schmued and Fallon, 1986, Brain Research 377:147-154). Since Fluoro-Gold is stable in many solvents and remains fast within retrogradely labeled neurons, it's use is compatible with many histochemical techniques. It can be used with other retrograde tracers, immunofluorescence, PAP and ABC immunocytochemistry, HRP histochemistry, autoradiography, counterstains (ethidium bromide is the preferred fluorescent counterstain), paraffin embedding and plastic embedding. However, if tissue is unfixed, additional processing of tissue in aqueous solutions for over an hour or two will result in loss of Fluoro-Gold fluorescence from labeled neurons. Fluoro-Gold may be useful in electron microscopy. Fluoro-Gold can be used in a brain which has been sectioned and transferred to phosphate buffer. Sections are typically mounted on gelatin-coated slides, air dried, immersed in xylene and coverslipped with DPX plastic mounting media (FLUKA Chemical Corp., 255 Oser Avenue, Hauppauge, New York, 11788, Catalog #44581). Tissue may also be viewed on slides without further processing, can be run through graded alcohols for dehydration, or, for immunocytochemistry, the sections can be air dried and directly coverslipped with neutral buffered glycerine (1:2).

Examination and Photography
Fluoro-Gold is visualized with a fluorescence microscope using a wide band ultraviolet (UV) excitation filter. Use the same filter pack you would for other fluorescent retrograde tracers excited under wide band UV (e.g., True Blue, Fast Blue, Nuclear Yellow), such as the Leitz Ploem filter system A (Wide Band UV, Excitation filter BP 340-380), Mirror RKP 400, Barrier Filter LP 430). Objectives should be made especially for fluorescence microscopy (such as that made by Zeiss) glycerine, or water. Since plastic does absorb UV light, it is not advised to view through plastic petri dishes, etc. Recommended films are T-Max (Kodak, black & white) and Ektachrome 200 (Kodak, color slides). Exposure times usually vary from 20 seconds to 1.5 minutes.

Chemical Analysis

Quality	Expected Result	Actual Result
Appearance	A golden-yellow, hygroscopic, crystalline powder	A bright-yellow powder
Odor	None	None
Solution	20 ml of a 5% w/v aqueous solution should be clean, clear and almost free from suspended matter, and should have not more than a very slight odor	Passes Test
pH of a 1% Solution	Between 4.0 and 5.5 at 25 degrees Celsius	4.6
Spectral characteristics	The spectral characteristics of Fluoro-Gold vary with pH	A 0.1% solution in distilled water has a pH of 4.5 and excitation peak of 414 nm and emission peak of 541 nm Fluoro-Gold bound to membranes at a physiological pH of 7.4 has an excitation band of 350 to 395 nm and an emission band of 530 to 600 nm
Chloride	Not more than 0.035%	0.017%
Sulfate	Not more than 0.1%	Less than 0.05%
Sulfated ash	Not more than 0.1%	Negligible
Heavy metals	Not more than 10 p.p.m	Less than 10 p.p.m
Selenium	Not more than 30 p.p.m	Less than 10 p.p.m.
Loss on drying	Not more than 1.0% after 3 hours in vacuo at 60 degrees Celsius	0.1%
Assay	Between 95.0 and 105.0% calculated with reference to the dried material	99.2%

Notice: The original and only true Fluoro-Gold (Fluorogold) is produced by Fluorochrome, LLC and marketed by Fluorochrome, LLC and Histo-Chem Inc.

Fluoro-Gold (Fluorogold) is an exclusive product of Fluorochrome, LLC. It has been sold by Fluorochrome and widely used since 1985. Other companies are marketing a product they claim is the same as or equivalent to Fluoro-Gold. In fact, the chemical structures of these compounds seem to be different from Fluoro-Gold. Certain physical properties of the compounds may be very different.

*CAUTION: Fluoro-Gold, Antibody to Fluoro-Gold and Fluoro-Ruby are for investigational use only in laboratory research animals or for tests in vitro. NOT FOR USE IN HUMANS. These drugs should be used only by persons regularly engaged in conducting neuroanatomical studies and tests in vitro or in animals used only for laboratory research.

貨號	品名	包裝	品牌
78000	熒光金 Fluoro-Gold	20mg	fluorochrome

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