WEKO3
アイテム
イヌおよびネコの表層角膜移植に関する研究
https://az.repo.nii.ac.jp/records/3229
https://az.repo.nii.ac.jp/records/32299dc87c14-fbc9-4de7-96f4-26d2b6e21343
| 名前 / ファイル | ライセンス | アクション |
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diss_dv_otsu0364 (12.0 MB)
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diss_dv_otsu0364_jab&rev (297.4 kB)
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diss_dv_otsu0364_eab.pdf (303.2 kB)
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| Item type | 学位論文 / Thesis or Dissertation(1) | |||||
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| 公開日 | 2013-02-05 | |||||
| タイトル | ||||||
| タイトル | イヌおよびネコの表層角膜移植に関する研究 | |||||
| タイトル | ||||||
| タイトル | Study of lamellar corneal transplantation in dogs and cats | |||||
| 言語 | en | |||||
| 言語 | ||||||
| 言語 | jpn | |||||
| 資源タイプ | ||||||
| 資源タイプ識別子 | http://purl.org/coar/resource_type/c_46ec | |||||
| 資源タイプ | thesis | |||||
| 著者 |
工藤, 莊六
× 工藤, 莊六× Kudo, Soroku |
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| 抄録 | ||||||
| 内容記述タイプ | Abstract | |||||
| 内容記述 | 小動物臨床における角膜の外科療法は、角膜病巣部の被膜処理により眼球を保全することに加えて、病巣角膜の透明性を回復し、視力を回復させるための処理として応用することが望まれている。 筆者は、この要望に応えてイヌおよびネコに対して同種の表層角膜移植を行った。しかし、近年、動物愛護の観点からイヌおよびネコの角膜採取には多くの制約があり、その入手は容易ではない。そこで、その代替えとして食用動物であるブタの角膜のイヌへの移植を検討した。試験に先立って、角膜病巣部の切除、移植片の作成、治療経過の観察などに必要な角膜の厚さ、および術後の局所保護に用いるコンタクトレンズの装着に必要な角膜曲率半径の測定を行った後、異種移植の試験を行った。 1.同種の表層角膜移植 イヌおよびネコに対してグリセリン保存した同種の角膜を用いて表層角膜移植を試みた。まず、基礎試験として健康なイヌ3頭3眼、ネコ2頭2眼に移植を試み、次いで臨床試験として角膜混濁を呈するイヌ8頭8眼、ネコ13頭13眼の臨床例に移植を実施し、角膜病巣の処置法としての臨床上の有用性を検討した。 移植に用いる保存角膜の作成は、飼い主から眼球提供の承諾を得た死後のイヌおよびネコの眼球を摘出し、抗菌剤溶液(2%アミノベンジルペニシリン溶液)で洗浄後、角膜周囲に強膜を2mm程度付着させて切除し、再度同抗菌剤溶液で洗浄し、滅菌したグリセリンに浸漬したまま4～6℃で6カ月～2年半冷蔵保存した。 角膜の使用に際して、グリセリンから取り出した強角膜片を100mlの生理食塩液に浸し、20分ごとに3回生理食塩液を取り替え、グリセリンを洗浄した。 移植片の作成は、ガーゼで角膜表面を擦過して角膜上皮を除去し、トレパンを用いて移植床と同じ大きさ、深さに切開し、ゴルフ刀で円周に沿って水平に切り込み、デスメ膜と内皮を残し、固有層のみを採取して移植片とした。 手術は、全身麻酔の後、手術用顕微鏡下で行った。まず、開瞼器を装着し、眼球結膜に2～4本の支持糸をかけ、眼球を固定した。必要な直径のトレパンを選び、切除する深さを設定し、トレパンの刃にフルオレセインを少量(約0.02ml)付着させ、角膜の円形切開を行った。フルオレセインで染色された切開部の一部を鑷子でつまみ上げ、ここからゴルフ刀で円周に沿って切除し、移植床とした。 切り出した移植片を移植床の上に載せ、4ヵ所を仮縫合した後、全周を連続縫合した。4本の仮縫合糸を取り除き、抗菌剤(リンコマイシン300mg/ml 0.3ml)の結膜下注射を行い、治療用コンタクトレンズの装着、あるいは眼瞼縫合によって眼球を被覆し手術を終えた。 術後はエリザベスカラーを装着し、広域スペクトルの抗菌剤の内服(オフロキサシン5mg/kg、1日2回)と点眼(0.3%オフロキサシン点眼液、1日3回)、およびコルチコステロイド(プレドニゾロン0.5mg/kg)の皮下注射、または経口投与を10～14日間行い、角膜上皮が移植片上を覆った後はコルチコステロイド(0.02%フルオロメトロン点眼液、1日3回)の点眼を2週間続けた。 眼瞼縫合は1～2週で解除し、角膜の縫合糸は2～6ヵ月で抜糸した。 その結果、基礎試験として正常な角膜への移植を行った5例では、角膜混濁、血管新生が見られたが、ほぼ1ヵ月で角膜移植片、母角膜共に透明となり全例生着した。 以上の基礎試験の成績に基づいて臨床試験を行った。すなわち、イヌ表層性角膜変性および表在性瘢痕、ネコ角膜分離症などの臨床例21例に本術式を応用した移植では、角膜中央部に及んだ病巣への移植片は、ほとんどの症例で初診時より透明度を増し、非常に良好11例、良好7例、不良2例、不明1例の好成績を得、臨床的に有用性の高いことを確認した。 2.角膜厚の測定 移植術を実施して、移植床の深さは個々の症例により差のあること、また、治癒の過程でその厚さに大きな経時的変化のあることが観察され、角膜の厚さの測定は、手術時に必要な検査の一つと思われた。そこで、正常な角膜の厚さを知るために健康なイヌ53頭106眼の角膜厚を超音波法によるパキメーターを用いて測定した。 その結果、イヌの生体では、角膜辺縁部の背側が0.636±0.59mmで最も厚く、外側および腹側、次いで内側の順で薄く、中心部の厚さが0.583±0.05mmで最も薄いことが観察された。 3.角膜曲率半径の測定 術後の角膜の庇護措置として、ヒトのコンタクトレンズ装着を試みたが、いずれも短時日で脱落し装着できず、角膜曲率半径を調査することが必要であった。そこで、健康なイヌ45頭90眼の角膜曲率半径の測定をオフサルモメーターを用いて行った。 その結果、角膜曲率半径は左右の差がなく、垂直方向で8.49±0.19㎜、水平方向で8.58±0.18mmであった。そして、体重と角膜曲率半径の間には、垂直方向、水平方向ともにr=0.58の有意相関(P＜0.01)が見られた。これを応用して、動物用コンタクトレンズの各サイズに臨床的に使用可能な体重を設定することができた。 4.異種の表層角膜移植 同種角膜の代替えとしてグリセリンに保存したブタ角膜を用い、表層角膜移植を健康なイヌ6頭6眼に試み、臨床応用への問題点と有用性を検討した。 まず、第一試験として3頭3眼に異種移植を試みた。保存角膜(第一試験75日、第二試験150日、4℃保存)の作成、術式は前述の同種移植と同様に行った。ただし、問題点を指摘するために、術後はコルチコステロイドによる消炎処置を行わず、同種移植例と同様の抗菌剤の点眼と内服のみを行い3週間観察した。日数の経過と共に移植片の膨化・白濁、母角膜の肥厚・混濁・血管新生、さらには肉芽の形成が見られた。 そこで、3週から8週目までコルチステロイド(0.02%フルオロメトロン点眼液、1日3回、3週間、その後0.25%酢酸プレドニゾロン眼軟膏、1日2回、2週間)の点眼を行った。 その結果、症状の改善が見られ移植片は生着した。しかし、1例は5週に入っても肉芽の形成が著しく消退しないので、外科的に肉芽の切除を行った。6週に入って母角膜は3例共に透明になったが、血管は1例のみゴースト血管となり、2例では消退せず、移植片は1例のみ透明、1例は半透明、1例は白濁で、後者の2例は充分な透明度を得ることが出来なかった。 次いで、第二試験として3頭3眼に異種移植を試みた。第一試験の経過を参考にして第二試験ではコルチコステロイドの使用を1週早め、2週から8週目まで点眼(0.02%フルオロメトロン点眼液、1日3回、4週間、その後0.25%酢酸プレドニゾロン眼軟膏、1日2回、2週間)を行った。その結果、移植片の生着・新生血管のゴースト化・肉芽の消退・母角膜の透明化は全例に見られ、移植片は1例で半透明(部分的)、2例で透明となり、全例に視力の回復が認められた。 これら両試験6例の術後1週における移植片周囲の角膜厚は、いずれも術前に比べ1.3～1.5倍に増加したが、術後3週以降、角膜厚は漸減することが確認された。母角膜厚の増加と共に血管新生が起こり、血管の周囲に混濁が生じ、これにともなって肉芽が発生するという一連の過程を観察することができた。これは、コルチコステロイドの投与時期、投与法を考慮する上で参考となった。 また、フォトケラトスコープによる角膜投影像(マイヤー像)は、術後1～3週で移植片上の投影同心円は非常に乱れ、角膜の歪みが認められたが、術後8週では第一試験の3例中1例および、第二試験の3例の全例に移植片上の同心円が明確に観察され、角膜の歪みが改善された。 以上の同種移植および異種移植の成績から、表層移植における問題点に対する解決法を導出した。すなわち、(1)移植片の作成に際しては、外層の角膜上皮および内層のデスメ膜、内皮を除き、角膜固有層のみを用いることにより、内皮型の拒絶反応を回避すること(2)移植床と移植片の周囲を確実に接着させ、角膜上皮の再生を促進させること(3)術後は直ちに抗菌剤ならびにコルチコステロイドの全身投与を継続し、角膜厚の増加と血管新生を防止すること(4)角膜上皮が移植片を被覆した後は、コルチコステロイドの点眼を継続し、移植角膜の透明度の増進をはかることなどである。 この様に本研究の成果は、イヌにおけるブタ角膜を用いた異種表層角膜移植が臨床に応用し得る新しい技術として期待できることを確認した。 |
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| Abstract | ||||||
| 内容記述タイプ | Other | |||||
| 内容記述 | Surgical corneal therapy is expected to be applied in the clinical practice for the treatment of small animals not only to preserve eye function by covering corneal lesions, but to improve the clarity of the injured cornea and consequently vision improvment. In this experiment, I have performed numerous lamellar corneal allotransplantation to facilitate and improve the procedure to meet the needs stated above. However, the animal rights movement has imposed severe limitations and made it more difficult to secure corneas from dogs and cats needed for these experimental studies. Therefore, as a transplant substitute, esculent porcine cornea were transplanted in to dogs and cats to evaluate modify and improve this experrimental procedure. First the thickness of the each cornea was measured, which is necessary for the successful resection of the corneal lesion, creating grafts and facilitates in the observation and evaluations of long-term follow-up of these experimental dog and cat allotransplants. Next the radius of the curvature of the cornea was determined, which is a necessary step prion to the application of contact lenses to cover the surgical area. 1. Lamellar Corneal Allotransplantation Lamellar corneal transplantation was performed on both dogs and cats utilizing allografts that has been preserved in glycerine. In the first phase, this basic experimental procedure was performed on three healthy dogs with normal eyes and two healthy cats with normal eyes. In the second phase, this procedure was performed, as part of a clinical study, on healthy dogs and thirteen healthy cats. One eye from each of the experimental animals was allotransplanted and all of these animals initially presented with exciting corneal opacity. This phase was designed to study and evaluate this transplantation procedure efficacy. The corneal grafts were prepared using the following procedual steps. The eyeballs were enucleated from cadaveric dogs and cats after I had obtained written informed consent from the owners, deceased animals donating these organs for this experimentation. Initially these eyes were rinsed thoroughly with antibiotic solution containing 2% aminobenzyl penicillin. The cornea was then resected to obtained a sclera of 2 mm width. The resected corneas were again rinsed with the same antibiotic solution and they were preserved in sterile glycerine at from 4° to 6℃, for period of from six months to two-and-a-half years. The preparation for the surgical transplantation of the corneal graft began with the soaking of each of the grafts in 100 ml of normal saline. This solution was replaced three times, following twenty minute intervals, to facilitate the washing out of the glycerine preservative from these grafts. Next the anterior epithelium of the cornea was removed by rubbing the corneal surface with sterile gauze and the graft was trimmed to using a trephine fit in the corneal bed. The lamina propria was resected by horizontally cutting along the circular edge utilizing a golf-club knife to remove Descemet's membrane and the endothelium. The operation was performed with the experimental animals under general anesthesia and using a surgical microscope. Eyelid speculum was applied and the operating field was obtained by the application of two to four threads through the conjunctival bulbi. The cornea was resected circularly using a trephine and applying 0.02 ml of fluorescein on its blade after determining the depth of the resection. The incision line, which was stained with fluorescein, was grasped with forceps and incised along the circular line with a golf-club knife. The corneal graft was applied on the corneal bed and fixed at four points with stay sutures then the circumference was continuously sutured and the four stay sutures were removed. Lincomycin (0.3 ml of 300 mg/ml) solution was injected subconjunctivally. Next therapeutic contact lens were applied over or the eyelids were sutured to cover the post surgical eyeball for our experimental sobject. An Elizabeth collar was applied to all animal subjects postoperatively. A broadspectrum antibiotic, Ofloxacin, was orally administered twice daily (5mg/kg) and was also instilled into the eyes in an ophthalmic solution (0.3 %). Also 0.5mg/kg of prednisolone, an anti-inflammatory corticosteroid, was injected subcutaneously or given orally for ten to fourteen days, until the corneal epithelium had been covered by the graft. Fluorometrone (0.02 %), a corticosteroid ophthalmic solution, was applied three times daily for two weeks. The eyelid sutures were removed one to two weeks post-operatively and the corneal sutures were removed after two to six months. In the five experimental cases that composed the first phase or the basic experiment which consisted transplantation of normal, healthy eyes, the transplanted eyes displayed transitional corneal opacity and neovascularization. However, both the graft and corneal bed cleared up within the first month post-operatively. No rejection reactions were encountered. The second phase was a clinical study where corneal allotransplantation was performed on a single eye dogs and cats that presented with corneal leisions. These initial conditions included canine superficial corneal dystrophy, superficial corneal scaring and feline corneal sequestration. The grafted cornea generally improved in clarity following the operation. The results showed eleven cases with excellent recovery, seven cases with good recovery, two cases of poor recovery and an unknown case. These results support the contention that this procedure is feasible and appropriate in this type of clinical setting. 2. Measurement of Corneal Thickness The thickness of the corneal bed tended to vary significantly with each case of corneal transplant and this thickness variation led to a wide variation in post-operative corneal thickness. Thus overall thickness appeared to be significantly influenced by the stage of the healing process, and it is thought that the measurement of corneal thickness may prove to be an important indicator for used pre-operative evaluation. Therefore, the corneal thickness was ultrasonically measured using a pachymeter for both eyes of 53 healthy normal dogs to establish a normal standard. The results indicate that the thickest portion of the cornea was the dorsal part of the corneal margin, which measured 0.636 ± 0.59 mm, followed by the lateral, ventral and medial portions, and the thinnest portion was at the center of the cornea which measured 0.583 ± 0.05 mm. 3. Measurement of the Corneal Curvature Radius Contact lenses, designed for human use, were applied to cover the dog and cat eyes post-operatively. However, this technique procedure very poor results, as these lenses tended to fall out after a period of several hours to a few days. This development led to the decision to measure the corneal radius curvature of both eyes from 45 dogs using an ophthalmometer. The results obtained had a mean curvature radius of 8.49 ± 0.19 mm along the vertical axis and 8.58 ± 0.18 mm along the horizontal axis. There were no detectable variation between the right and the left eyes. A positive correlation (r=0.58, p<0.0l) was discovered between the weight and the corneal curvature radius along both the vertical and horizontal axes. This result can be applied to determine the appropriate contact lens size for the experrimental animals based upon the specific weight of each of the animals. 4. Lamellar Corneal Heterotransplant The glycerine-preserved porcine corneal grafts were used for the allotransplantation of lamellar corneal transplants into six healthy eyes of six dogs, to evaluate the clinical applicability of this procedure. The first experimental surgery was performed on three single eyes of three dogs. The grafts were prepared and preserved (75 days for the first experiments and 150 days for the second, at 4 °C). The operation was performed utilizing the same procedure as for the allotransplant guidelines. However, the anti-inflammatory therapy was not carried out to identify problem subjects. These experimental canine subjects were treated with oral antibiotics and antibiotic eye drops for three weeks along the same protocol as for allotransplants. Graft swelling and opacification, corneal bed thickning and opacification, neovascularization and granulation formation were observed post-operatively. Three weeks after the operation, 0.02 % fluorometrone ophthalmic solution was administered three times daily over a period of three weeks. Then, 0.25 % prednisolone succinate eye ointment was applied twice daily for two weeks. In cases in which the granulation did not regress after five weeks of treatment surgical resection of this granulation. The corneal beds clarified six weeks post-operatively in all the three cases. However, there was one case with ghost vessels, while the rest did not show any regression of neovascularizated vessels. One graft was clear, one was partially clear while the other was opaque. Therefore, two out of three did not gain clarity. The second experiment was further modified and improved based on the experience and results gained from the first experiment. In the second experiment, the corticosteroid was initiated a week earlier than for the first experiment. Also 0.02 % fluorometrone ophthalmic solution was initiated two weeks post-operatively and applied three times daily for a three weeks period. Prednisolone 0.25 % was applied succinate two times daily for a two-week period. As a result, all three cases exhibited live grafting, neo-vessel ghosts, regression in granulation, and clarity in the corneal bed. The graft was partially clear in one case, while the other two had clear grafts. All three animals regained their eyesight. In the one-week post-operative period, these six corneas became 1.3 to 1.5 times thicker than they were in their pre-operative state. This observed increase in corneal thickness gradually decreased during first three weeks post-operative. The series of processes, including thickening of corneal bed with neovascularization and opacity around neo-vessels followed by granulation formation, were observed. They are proposed as markers or factors for the evaluation and detarmination for the appropriate timing and dosage of the corticosteroids. Corneal projection using a photokeratoscope demonstrated that one-to-three week post-operative grafts were shaped like a significantly irregular concentric circle with a distorted cornea. However, by eight-weeks post-operative, one of three animals from the first experiment and all three eyes from the second experiment group exhibited grafts with regular concentric circles in which the earlier distortion had been corrected. The problems I encountered in the allotransplantation and heterotransplantation experimental procedures were solved with the following modifications of the protocol: 1. Rejection reaction can be avoided by using as a graft only the lamina propria, after the removal of the anterior epithelium, Descemet's membrane and the endothelium. 2. Promotion of the regeneration of the corneal epithelium is necessary, by securely suturing the graft to the corneal bed. 3. Systemic administration of corticosteroids started immediately after the operation can prevent the increased thickness of the cornea and neovascularization. 4. Corticosteroid ophthalmic solution should be continuously after the corneal epithelium covers the graft, to improve the clarity of the grafted cornea. In conclusion, the series of the current experiments suggest that the lamellar corneal heterotransplant using porcine corneal graft can be clinically applied to dogs with success. |
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| 学位名 | ||||||
| 学位名 | 博士(獣医学) | |||||
| 学位授与機関 | ||||||
| 学位授与機関名 | 麻布大学 | |||||
| 学位授与年月日 | ||||||
| 学位授与年月日 | 1997-12-03 | |||||
| 学位授与番号 | ||||||
| 学位授与番号 | 乙第364号 | |||||
| 著者版フラグ | ||||||
| 出版タイプ | AM | |||||
| 出版タイプResource | http://purl.org/coar/version/c_ab4af688f83e57aa | |||||
