EXOTIC ANIMAL ENDOSCOPY

EXOTIC ANIMAL ENDOSCOPY

in association with

Karl Storz Endoscopy

Brno/CZ, September 2008

DR STEPHEN J. HERNANDEZ-DIVERS

BVETMED, CBIOL MIBIOL, MRCVS, DZOOMED(REPTILIAN), DIPL. ACZM, RCVS SPECIALIST IN ZOO & WILDLIFE MEDICINE

ASSOCIATE PROFESSOR OF EXOTIC ANIMAL, WILDLIFE AND ZOOLOGICAL MEDICINE, DEPARTMENT OF SMALL ANIMAL MEDICINE & SURGERY,

COLLEGE OF VETERINARY MEDICINE, UNIVERSITY OF GEORGIA,

ATHENS, GEORGIA 30602-7390 TEL: 1-706-542-6378 FAX: 1-706-542-6460

EMAIL: [email protected]

INTRODUCTION

Endoscopy has proven to be a useful diagnostic tool inveterinary medicine (Tams, 1999, McCarthy, 2005). In thefield of zoological medicine, the application of diagnosticendoscopy has shown promise in many taxa. It has proba-bly been most successfully employed in avian speciesthanks to their unique airsac system that facilitates coe-lioscopy without insufflation (Harrison, 1978, Taylor,1994, Hernandez-Divers and Hernandez-Divers, 2004).Reptile endoscopy has not enjoyed such widespreadacclaim although the simple coelomic design of mostspecies makes them ideally suited to coelioscopy. Themajority of previous reports of reptile endoscopy describeforeign body retrieval from the gastrointestinal tract(Lumeij and Happe, 1985, Ackermann and Carpenter,1995). Descriptions of coelioscopy (including sex identifi-cation, cloacoscopy, tracheobronchoscopy, and otherclinically relevant techniques, particularly in cheloniansalso exist (Coppoolse and Zwart, 1985, Cooper, 1991,Schildger and Wicker, 1992, Gobel and Jurina, 1994,Burrows and Heard, 1999, Divers, 1999, Schildger, et al,1999, Hernandez-Divers, 2001, Hernandez-Divers andShearer, 2002, Hernandez-Divers, 2004, Hernandez-Divers, et al, 2004a).

Clinical, research and teaching experience suggests thatendoscopy offers unparalleled opportunities for visualiza-tion and biopsy in reptiles, and has been advocated as a

standard diagnostic technique (Schildger and Wicker,1992, Schildger, 1994, Divers, 1999, Schildger, et al,1999, Hernandez-Divers, 2003, 2004, Hernandez-Divers,et al, 2004a, Hernandez-Divers, et al, 2004b). A standardsingle-entry system incorporates a rigid telescope housedwithin a sheath, through which basic instruments can beinserted into the field of view. For reptiles over 10 kg,larger sheathed telescopes, separate cannulae, and instru-ments are used by triangulation (Magne and Tams, 1999,McCarthy, 2005). This paper reviews diagnostic coe-lioscopy in reptiles, and describes techniques that havebeen efficacious in a variety of squamates, crocodilians,and chelonians.

Rigid endoscopy equipment — Older rigid endoscopesincorporated a convex glass lens system, in which smallglass lenses were separated by large air spaces. By con-trast, modern telescopes incorporate a rod lens design thatutilizes comparatively longer rods of glass and smaller airspaces. The advantages of the rod lens system are greaterlight transmission, better image resolution, wider field ofview and image magnification. The general concensusamong endoscopists is that rod lens telescopes are superi-or. The authors use the system designed by ProfessorHarold Hopkins and manufactured by Karl Storz (KarlStorz Veterinary Endoscopy America Inc, Goleta, CA)(Table 1). Human cystoscopy equipment (2.7 mm) wasadopted for avian coelioscopy and is equally applicable to

Volume 15, No. 3, 2005 Journal of Herpetological Medicine and Surgery 41

A Review of Reptile Diagnostic Coelioscopy

Stephen J. Hernandez-Divers1, BVetMed, DZooMed (Reptilian), MRCVS, DACZM, Sonia M. Hernandez-Divers1, DVM, DACZM,

Heather G. Wilson1, DVM, DABVP-Av, Scott J. Stahl2, DVM, DABVP-Av

1. Exotic Animal, Wildlife and Zoological Medicine, Department of Small Animal Medicine & Surgery,

College of Veterinary Medicine, University of Georgia, Athens, GA 30602, USA

2. Stahl Exotic Animal Veterinary Services, 111A Center Street South, Vienna, VA 22180, USA

ABSTRACT: Diagnostic endoscopy has proven to be an important diagnostic tool for minimally-invasivevisualization and biopsy of internal structures in a variety of species. In reptile medicine, the lack ofpathognomonic clinical signs, variable hematology and inconsistent plasma biochemistry results, makedisease diagnosis challenging. In many cases reaching a definitive diagnosis relies upon biopsies for his-tology and microbiology. The ability to explore the coelom and collect biologic samples with targetedprecision and minimal trauma using a small diameter rigid telescope with intergrated sheath and operat-ing channel offers a significant diagnostic advantage over surgical coeliotomy or ultrasound-guidedtechniques. This review describes available equipment, approaches and techniques for examination ofcoelomic viscera for members of the Squamata, Crocodilia and Chelonia. Emphasis is placed upon the2.7 mm telescope system as this size is suitable for the majority of reptile species; however, 5 and 10 mmequipment for larger species is also described.

KEY WORDS: reptile, surgery, endoscopy, coelioscopy, diagnosis, biopsy.

T E C H N I Q U E S

42 Journal of Herpetological Medicine and Surgery Volume 15, No. 3, 2005

most reptiles under 10 kg. In contrast to a 0° telescope, the30° Hopkins telescope not only enables a straight aheadview but, by rotating the scope around its longitudinalaxis, a greater area can be surveyed. The 2.7 mm telescopecan be housed within an examination and protection sheathor an operating sheath with an intergrated instrument chan-nel. The operating sheath provides two stop-cocks for usessuch as insufflation, aspiration and irrigation and an oper-ating channel that accommodates various instruments,including scissors, grasping forceps, biopsy forceps, fineaspiration/injection needle, wire-basket snare, laser, andradiosurgical probes (Figure 1). The biopsy forceps areused to harvest tissue samples for histopathology andmicrobiology. The small sample size permits the collectionof biopsies for various laboratory tests, and for sequentialbiopsies over time to monitor disease progress. Grasping

forceps (5 Fr) are useful for manipulating tissues, debride-ment, and foreign body removal. The fineaspiration/injection needle is used for aspiration, irrigationand drug administration. A smaller 1.9 mm telescope ver-sion is available with 3 Fr grasping and biopsyinstruments, and is ideally suited to animals less than 100g(Table 1).

For larger reptiles, over 5 – 10 kg, cannulae are used tocreate multiple ports for the insertion of telescopes andinstruments (Magne and Tams, 1999, McCarthy, 2005)(Figures 2 and 3). The endotip cannula is a recentimprovement that has an external screw-thread to enablegradual advancement by rotation (Figures 2 and 3)(Ternamian and Deitel, 1999) The cannula does not requiretrocar or axial penetration force during insertion. A tele-scope within the cannula provides a magnified view duringentry into the coelom. As the cannula is advanced througha small skin incision, the fascia and then the muscle fibersspread radially and are transposed onto the cannula’s outerthread. The thin pleuroperitoneal membrane is transillumi-nated so that viscera, vessels and/or adhesions arevisualized before entry into the coelom. The risks of iatro-genic visceral damage are therefore greatly reduced. A 5mm endotip cannula can be used with a 2.7 mm telescopesheathed within a 3.5 mm protection sheath, while 5 and10 mm telescopes and instruments can be inserted through6 and 11 mm endotip cannulae respectively. Telescopesand instruments of the same size can be used interchange-able between multiple endotip ports.

For reptiles between 5 and 100 kg, 5 mm telescopes andinstruments are used, but for animals over 100 kg, 10 mmequipment may be preferred. Various 5 and 10 mm instru-ments that accompany the different diameters of cannulaeare available for performing endoscopic surgery. However,for the purposes of this review, diagnostic instruments willbe discussed (Figure 3).

There are two types of light source available, tungsten-halogen and rare-earth xenon, and either is connected tothe telescope via a flexible, fiber-optic cable. Halogen issufficient for rigid endoscopy using the eye-piece in smallanimals. However, xenon is generally preferred becausethe greater intensity and quality of light provides betterreal-life and recorded images. Xenon becomes more bene-ficial as the telescope diameter decreases or the patientsize increases above 1 kg. A xenon light source with a ded-icated endoscopy camera and a recording device (e.g.analogue video, digital video, digital still image capture,still image print-out) is recommended for wide speciesapplication, case records, and client education (Figure 4).Cameras that relay the endoscopic image from the eye-piece to a monitor (endovideo cameras) were onceconsidered optional but clinical, research and trainingexperience has indicated that surgeon ergonomics and abil-ity are greatly improved by their routine use (Figures 1Aand 2C).

Insufflation is essential for reptile coelioscopy in orderto create the necessary working space. Several gases havebeen used, but medical grade CO2 is inert, non-toxic, read-ily absorbed, quickly excreted, and is preferred. DedicatedCO2 endoflators accurately control gas flow to maintainthe desired insufflation pressure; however, it is possible to

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Table 1. Rigid Endoscopy Equipment Used for ReptileDiagnostic Coelioscopy


use a simple aquarium air pump to provide room air forinsufflation.

Operating room design and layout are important. Thelight source, camera unit, endoflator, and documentationdevice are best stored on a mobile cart that can be easilymoved and positioned in the operating room (Figure 5A).An endovideo camera coupled to a monitor facing the sur-geon at eye-level will greatly improve the ability of theendoscopist and reduce fatigue. Standard surgical and

endoscopic equipment and supplies should also bearranged within easy reach (Figure 5B). After the equip-ment is cleaned using a neutral pH enzymatic cleaner, itcan be sterilized using hydrogen peroxide vapor or ethyl-ene oxide gas. Cold sterilization using glutaraldehydeaccording to the recommendations of the manufacturer isacceptable. Autoclaving has not been routinely advisedbecause of fears of reduced equipment longevity; however,most modern telescopes are autoclavable.

Patient preparation — For elective procedures, reptilesshould be fasted to reduce the volume of the gastrointesti-nal tract. The precise duration of fasting will depend uponspecies, age, and dietary preferences. In those reptiles thatpossess a urinary bladder, digital stimulation of the cloacaprior to anesthetic induction may promote urination andreduce the size of the bladder, thereby reducing thechances of iatrogenic trauma during telescope entry intothe coelom. Alternatively, urinary catheterization and emp-tying of the bladder may be possible.

Laparoscopy and CO2 insufflation are known to bepainful procedures that necessitate general anesthesia inhumans (Golditch, 1971, Kehlet, 1999). Therefore generalanesthesia and analgesia are considered equally essentialfor reptile coelioscopy. Insufflation causes visceral dis-placement and places tension on visceral suspensoryligaments, making sedation and/or local anesthesia of theentry site inadequate for clinical practice. Following theinduction of general anesthesia, tracheal intubation andartificial ventilation are essential to overcome apnea andthe adverse effects of insufflation on lung inflation.Insufflation gas must be evacuated from the coelom beforefinal closure to reduce post-operative discomfort.

Telescope, instrument and biopsy handling — A sheath,although increasing overall diameter, is recommended toavoid damage to the smaller 1.9 and 2.7 mm telescopes,and in most cases an operating sheath is preferred to allow

Figure 1. Basic rigid endoscopy system. (A) 2.7 mm telescopehoused within a 14.5 Fr operating sheath (1), insufflation/irriga-tion stop-cocks (2), operating channel (3), endovideo camera (4),and light guide cable (5); (B) close-up of the end of the operat-ing sheath illustrating biopsy forceps protruding from theinstrument channel (Courtesy of Karl Storz VeterinaryEndoscopy); (C) endoscopic instruments (5 Fr) for use with the14.5 Fr operating sheath - grasping forceps (1), biopsy forceps(2), aspiration/injection needle (3), and single-action scissors (4)(Courtesy of Karl Storz Veterinary Endoscopy).

Figure 2. Rigid 5 mm endoscopy equipment suitable for reptilesover 10 kg. (A) 5 mm telescope (1), 5 mm biopsy forceps (2),10.5 cm 6 mm endotip cannula (3), and 6.5 cm 6 mm endotipcannula (4); (B) 10.5 cm 6 mm endotip cannula with externalthread for screw insertion (1), multifunctional telescope andinstrument valve (2), and insufflation stop-cock (3).

Figure 3. Diagnostic coelioscopy through the prefemoral fossaof an adult Aldabra tortoise (Aldabrachelys gigantea). (A) insert-ing an endotip cannula under telescope guidance; (B) placementof two 6 mm endotip cannulae into the prefemoral fossa for tele-scope and instrument insertion; (C) examination of theinsufflated coelom (oviduct on monitor) using a 5 mm 30° tele-scope.


instrument use. When moving around the coelom, the sur-geon should sit facing the monitor at eye-level. Thesurgeon’s inferior hand is used to hold the shaft of thesheath, close to where it enters the coelom, while the dom-inant hand is used to support and control the main body ofthe telescope-sheath-camera unit (Figure 6a). This han-dling technique enables the endoscopist to maneuver thetelescope around the coelom with maximum control andminimal tremor. Before using an instrument, this two-handed grip must be modified. The inferior hand is used toform a fist around the shaft of the sheath with the thumbslid proximally to prevent rotation. This enables the inferi-or hand to take the weight and maintain the position of thetelescope-sheath-camera, while freeing the dominant handto manipulate an instrument into the operating channel ofthe sheath (Figure 6b). Consider the collection of a kidneybiopsy from a lizard (Figure 6). Once the instrument hasbeen inserted into the endoscopic field of view, it is impor-tant to move the instrument-telescope-sheath-camera as asingle unit when approaching the structure of interest.Independent movement of the instrument is not only moredifficult but often results in poor control. The biopsyinstruments are sharp and delicate, and it is not necessaryto forcibly close the forceps with great pressure. Thespring action of the handle is often sufficient, but lightassistance to close the jaws is all that is ever required.Excessive force will increase crush artifact, and risk instru-ment damage. In some situations, the membranes coveringan organ may be tough and incision using scissors mayimprove access for tissue collection (Figures 7 and 11).

Upon withdrawal of the biopsy instrument from theoperating sheath, the endoscopist opens the biopsy jawsand an assistant, using a moistened sterile cotton-tippedapplicator, gently rolls the biopsy onto the applicator. Thebiopsy is then transferred to a foam-sandwiched histologycassette, which is closed and placed into neutral bufferedformalin for histology. For microbiologic culture, the tis-sue biopsy can be placed either into transport medium or,if submitted immediately, a sterile blood tube containing asmall volume of sterile saline (not bacteriostatic water) toprevent tissue desiccation.

COELIOSCOPY - SAURIA AND CROCODILIAPatient positioning — Coelioscopy of the green iguanahas been objectively assessed and serves as a useful modelfor most saurians (Hernandez-Divers, et al, 2004a). Giventhe small size of most lizard species, entry in a paramedianor paralumbar area will permit examination of most, if notall, coelomic structures (Figure 8). For a left paralumbarapproach, the lizard is positioned in right lateral recumben-cy with the left hindlimb taped caudad against the tailbase. The surgical area is bordered by the ribs, spine andhindlimb, and a central paralumbar entry is standard.Small crocodilians are placed in dorsal recumbency for aventral paramedian approach because osteoderms makethe paralumbar approach more difficult.

Endoscopic procedure — The precise entry point will bedictated by diagnostic imaging, anatomic asymmetry ofthe coelomic viscera, and the preferences of the endo-scopist. Following aseptic preparation, a 2 – 4 mm skin

Figure 4. Sample endoscopy report that can be generated whenan endoscopy camera and digital documentation equipment areavailable.

Figure 5. Endoscopy room preparation. (A) Endoscopy towerwith all required equipment including monitor (1), digital docu-mentation and printer (2), camera base-unit (3), light-source (4),and CO2 endoflator (5); (B) operating room layout for cheloniancoelioscopy demonstrating the patient, equipment and surgeonposition.


incision is made in the middle of the paralumbar region.To avoid damage to visceral structures, the skin and under-lying musculature are pinched and elevated using thumband forefinger before the operating sheath and obturatorare forced through the thin coelomic wall (composed ofthe internal and external oblique muscles and pleuroperi-toneum) and into the coelom (Figure 9). Blunt penetrationtends to ensure an adequate seal and prevent insufflationgas leakage around the sheath. Alternatively, a surgicalcutdown procedure and dissection through the muscle lay-ers can be adopted as long as a pursestring suture is placedaround the sheath. A sheath stop-cock is connected to theCO2 endoflator and set to 0.4 – 0.7 KPa (3 – 5 mmHg).The obturator is removed and replaced with the telescope.When using an aquarium air pump for insufflation, thesecond sheath stop-cock is left open to avoid over-infla-tion. Air is permitted to continuously escape from thesystem because, unlike a dedicated endoflator, an aquari-um air pump cannot be set to control the gas flow tomaintain a precise insufflation pressure. Occluding thissecond stop-cock with a finger increases insufflation pres-sure, while lifting the finger off the stop-cock openingdecreases insufflation. By careful finger control, insuffla-tion can be crudely controlled. Alternatively, the secondstop-cock can be partially closed to balance the inflow andoutflow of gas.

Once the endoscope has been inserted, it is often neces-sary to gently touch the tip of the telescope against apleuroperitoneal membrane to clean the terminal lens ofcondensation or fluid. If there is fat or blood on the lens itis usually more effective to remove the telescope from thesheath, clean with sterile damp gauze, and then replace. Itis important not to continue with a dirty lens as poor visu-alization will reduce the endoscopist’s ability and increaseprocedure time.

Upon entry, the first organ to find for orientation is thelarge, brown liver lying in the mid-ventral coelom (Figures10 and 11). Advancing the telescope craniad will reveal theheart, and dorsad the lungs (Figure 12). There are nodiaphragmatic, post-pulmonary, or longitudinal mem-branes in most saurians. However, these membranes doexist to a greater or lesser extent in tegus, monitors, andcrocodilians (Perry, 1998). Minor perforation of thesemembranes with the telescope will not cause any signifi-cant harm as long as the lung, intestinal tract and bladderare not perforated. Dorsal to the heart and extending fromthe cranial coelomic inlet to mid-coelom are the pairedlungs. In most species the caudal lung becomes thin andsac-like, and in the Chameleonidae finger-like projectionsare observed. Although reptiles will tolerate hypoxia, amechanical ventilator is recommended to maintain lungventilation during coelioscopy. Lung ventilation will besubstantially reduced by insufflation and careful communi-cation with the anesthetist is required to balanceinspiration and insufflation pressures. Inspiration pressuremust exceed coelomic insufflation pressure for lungexpansion, and decrease below insufflation pressure forexpiration.

Caudal to the lungs, the stomach resides in the mid-coelom (Figure 13). The duodenum is biased towards theright side, in close association with the majority of thepancreas, while the ileum is more easily located on theleft, often caudoventral to the stomach. The large intestinecan often be appreciated from both sides but in hindgut

Figure 6. Correct handling of the 2.7 mm telescope system. (A)Two-handed technique illustrating control of the tip using theinferior hand, while supporting the sheathed telescope and cam-era with the superior hand. This technique maximizes finecontrol and reduces fatigue; (B) one-handed technique with theinferior hand supporting the shaft of the sheath and the weight ofthe camera, while the superior hand manipulates the instrument.This technique is only safe if the telescope is correctly housedwithin the sheath.


fermenting species like the green iguana (Iguana iguana)it is often displaced to the right.

The gonads are located in mid-coelom, on each side ofthe dorsal midline (Figure 14). Sex identification can bedetermined endoscopically at an early age, even inmonomorphic species of the genera Tiliqua, Corucia,Varanus, and Heloderma. Endoscopy also provides feed-back on gonadal activity and disease. The testes are ovoidand smooth and the immature or inactive ovaries appear assmall clusters of clear, fluid-filled, follicles. The gonadsmay enlarge tremendously during seasonal reproductiveactivity. The vasa deferentia of males and oviducts offemales can be followed caudad to the kidneys andurodeum, respectively. Depending upon species, kidneysmay be examined on each side of the dorsal midline in thecaudal coelom (Figure 15).

The spleen is closely associated with the greater curva-ture of the stomach, and in some species, carefulexamination ventromedial to the stomach and spleenreveals the splenic limb of the pancreas (Figure 16A and16B). The adrenal glands are dorsal to the gonads and liealong the renal veins, and the bladder, if present, is foundwithin the most dependent aspect of the caudal coelom,close to the caudoventral fat-body (Figure 16C and D). A

Figure 7. Instrument handling and biopsy technique. (A)Endoscopic view of an iguanid kidney; (B) endoscopic scissorsinserted down the instrument channel of the sheath into the fieldof view, and used to incise the renal capsule; (C) the scissors arewithdrawn and the incised capsule reveals the renal parenchymabelow; (D) biopsy forceps inserted into view and through thecapsular incision to collect a tissue sample.

Figure 8. Lizard positioning for coelioscopy. (A) Green iguana(Iguana iguana) in lateral recumbency with paralumbar regiondelineated and the preferred entry site marked (X); (B) paramedi-an entry (arrow) in a leopard gecko (Eublepharis macularius)with the position of the paired pelvic veins draining into the mid-line abdominal vein indicated.


Figure 9. Lizard coelisocopic technique with drapes removed forphotography. (A) Following aseptic preparation and using aseptictechniques, the skin and coelomic musculature is pinched andelevated using thumb and forefinger; (B) a 2- 4 mm incision inmade through the skin; (C) while holding the skin and muscle,the sheath and obturator are inserted through the skin incisionand gently forced into the coelom; (D) the CO2 line is connected(arrow) and, following insufflation, coelioscopy can commenceusing the two-handed technique. The endoscopy light visiblethrough the body wall can be helpful for orientation.

Figure 10. Lizard coelisocopy. (A) Right liver lobe (1) and gallbladder (2) in a green iguana (Iguana iguana); (B) left liver lobein green iguana, note the dark pigmented areas ofmelanomacrophage aggregation; (C) numerous pale foci withinthe liver of a veiled chameleon (Chameleo calyptratus) withmultifocal bacterial hepatitis; (D) hepatomegaly due to amyloi-dosis in a green iguana.

Figure 11. Endoscopic liver biopsy in lizards and chelonians(A) Caudal edge liver biopsy using 5 Fr biopsy forceps in agreen iguana (Iguana iguana); (B) note the the minimal hemor-rhage from the biopsy site (arrow); (C) incision through thepleuroperitoneal and hepatic membranes of an Egyptian tortoise(Testudo kleinmanni) in preparation for liver biopsy; (D) inser-tion of the biopsy forceps into the liver of the same Egyptiantortoise to collect a deeper parenchymal biopsy.

Figure 12. Lizard coelioscopy. (A) Heart (1) and deflated lung(2) in the cranial coelom in a green iguana (Iguana iguana); (B)Deflated lung (1) and spine (2) in a green iguana; (C) post-pul-monary membrane in an Iranian monitor (Varanus bengalensis);(D) multiple urate tophi on the lung surface of a green iguanasuffering from chronic renal disease.


right paralumbar approach will provide greater access tothe gall bladder at the caudal edge of the right liver lobe,while the pancreas is located adjacent to the duodenum(Figure 10A and 13B). In addition, the iguanid sacculatedcolon is more readily appreciated from the right side(Figure 13D).

Abnormal structures should be documented and samplescollected using biopsy forceps (Figure 17). When dealingwith potentially cystic structures, the fine aspiration needlereduces the risk of post-sampling leakage and contamina-tion of the coelom compared to biopsy forceps. Careshould be taken when collecting samples from the surfaceof the gastrointestinal or urogenital tracts as perforationmay result in leakage and coelomitis. In addition, bloodvessels and nerves should be avoided unless lesions arelarge and can be sampled without damaging the integrityof structure. Following coelioscopy, insufflation gas isaspirated followed by routine skin closure using a singlesuture and/or tissue adhesive (VetBond, 3M, St. Paul,MN). There is no need to repair the small puncture woundin the coelomic musculature.

COELIOSCOPY – SERPENTESPatient positioning — Coelioscopy in the snake is not asrewarding or straightforward as it is in the lizard. The elon-gated body design of the snake makes it impossible toexamine all organs from a single entry point. In addition,the more diffuse fat bodies, and numerous fascial planesmake insufflation and navigation more difficult. A targeted

Figure 13. Lizard coelioscopy. (A) Stomach in a green iguana(Iguana iguana) (arrow); (B) pancreas (1) lying adjacent to theduodenum (2) in a green iguana, note also the midline aB. den-drobatidis ominal vein (3) and gall bladder (4); (C) loops ofileum (arrow) seen from the left side in a green iguana; (D)large sacculated colon (arrow) seen from the right side in agreen iguana.

Figure 14. Lizard coelioscopy. (A) Testis (1), epididymis (2) andadrenal gland (3) in an adult green iguana (Iguana iguana); (B)vas deferens (arrow) coursing caudad towards the kidney in anadult male iguana; (C) ovary (1) and infundibulum (2) in a sub-adult female iguana; (D) involuted oviduct (arrow) in a femalesavannah monitor (Varanus exanthematicus).

Figure 15. Lizard coelioscopy. (A) Normal kidney (1) in a greeniguana (Iguana iguana) with vas deferens (2) and large intestine(3) in close association; (B) renal biopsy using 5 Fr biopsy for-ceps in a green iguana; (C) renomegaly in an iguana with chronicglomerulonephrosis; (D) bacterial infarcts (arrows) in the kidneyof a Yemen chameleon (Chameleo calyptratus).


coelioscopic approach can be used to examine and biopsyfrom a specific area, e.g. liver or kidneys. The preciseentry point is governed by species-specific anatomy(McCracken, 1999).

Endoscopic procedure — Snakes are placed in lateralrecumbency. In small snakes the telescope may be insertedbetween the first and second row of lateral scales to enterthe coelom ventromedial to the ribs. In larger specimens,the telescope can enter through the intercostal muscles,between two ribs. The difficulty of telescope introductionin snakes can be reduced with a Veress needle to inducepneumocoelom prior to sheath-obturator insertion, or by anoptical or endotip cannula that can be inserted under directvisual control (Ternamian and Deitel, 1999). The less dis-tendable coelom of snakes may also warrant increasedinsufflation pressures of up to 0.8 – 1.4 KPa (6 – 10mmHg) to create an adequate pneumocoelom. Targetedexamination and biopsy of liver, kidney, and splenopan-creas are possible (Figure 18). The rigid telescope can beused for lung examination via a temporary pneumotomy. Asmall coeliotomy approach is performed to identify thelung. While maintaining maximum inspiration a stab inci-sion is made into the lung and the telescope is inserted. Apurse-string suture is used to ensure an adequate seal andprevent the escape of anesthetic gas.

COELIOSCOPY – CHELONIAPatient positioning – The most useful endoscopicapproach to the chelonian coelom is through theprefemoral fossa. Unless, diagnostic imaging or anatomicconsiderations dictate otherwise, the decision about a leftor right approach can be determined by the preference ofthe endoscopist. The conformation of the shell andprefemoral fossa makes a left fossa approach easier forright-handed surgeons, and a right fossa approach easierfor left-handed surgeons.

The chelonian is positioned in lateral recumbency usinga vacuum positioning cushion (Vac-Pacs, OlympicMedical, Seattle, WA) or sand-bags. The pelvic limb isretracted and secured caudad to expose the prefemoralfossa (Figure 19). In chelonians with a pronounced caudalplastron hinge it is usually necessary to place a wedgebetween the caudal plastron and carapace to maintain ade-quate exposure of the prefemoral fossa.

Endoscopic procedure — Following aseptic preparationof the prefemoral area and surrounding shell, a 2 – 4 mmcranial to caudal skin incision is made in the center of thefossa. The subcutaneous fat and connective tissues arebluntly dissected using hemostats. This dissection is con-tinued to the level of the coelomic aponeurosis, which isformed by the broad tendinous portions of the transverseand oblique abominal muscles. Muscle damage and hem-

Figure 16. Lizard coelioscopy. (A) Elongated spleen (1) andclosely associated stomach (2), and testis (3) in a green iguana(Iguana iguana); (B) spherical spleen (1) and stomach (2) in aYemen chameleon (Chameleo calyptratus); (C) adrenal gland(arrow) dorsal to the epididymis (1) and testis (2) in a male igua-na; (D) urinary bladder in an iguana.

Figure 17. Lizard coelioscopy. (A) Vestigial yolk sac (arrow)attached via a short stalk to the intestinal tract (1) of a sub-adultgreen iguana (Iguana iguana); (B) two fungal granulomasattached to the pleuroperitoneal membrane of the coelomic wallin a green iguana (Iguana iguana); (C) hepatic cyst (arrow)attached to the liver (1) and close to the small intestine (2) in abearded dragon (Pogona vitticeps); (D) multifocal pale streakswithin the internal abdominal oblique muscle of an iguana withchronic renal disease, biopsy confirmed metastatic soft tissueminerlization.

orrhage can be avoided by remaining cranial and ventral tothe sartorius and iliacus muscles, respectively. Entry intothe coelom is accomplished by penetrating the coelomicaponeurosis with the sheath and obturator, aiming towardsthe mid-point of the cranial rim of the carapace (Figure20). Insufflation is essential and is provided using thesame techniques at the same pressures as previouslydescribed for lizards.

Identification of the prominent liver is used to orientatethe endoscopist (Figure 21). The stomach lies in a cran-iodorsal location, often partially obscured by the liver. Theintestinal tract may be viewed from either side, althoughthe duodenum is easier to locate from the right (Figure 22).The inactive ovary and oviduct are situated in the cau-dodorsal coelom but once mature may occupy much of thecentral region (Figure 23a–c). The male testis, oftencream, yellow or brown in color, epididymis, and vas def-erens are readily visible (Figure 23d). The adrenal glandslie craniomedial to the gonads and the retrocoelomic kid-neys are located in the caudodorsal coelom. Theretrocoelomic kidneys may be obscured behind a pigment-ed coelomic membrane making it impossible to identify orbiopsy unless this covering is incised and reflected (Figure24). The pancreas and spleen can be challenging to find,but are more commonly located on the right side (Figure25a,b). The lungs are situated dorsad and typically only theipsilateral lung is visualized from a lateral approach. In


Figure 18. Coelioscopic view of the kidney (1), fat body (2), ribsand intercostal muscles (3) in a boa constrictor (Boa constrictor).Note the relatively poor degree of coelomic expansion despiteinsufflation, which is common in snakes.

Figure 19. Chelonian positioning for coelioscopy. (A)Loggerhead sea turtle (Caretta caretta) placed in lateral positionwith the pelvic limb secured to expose the prefemoral fossa andtelescope entry site (arrow); (B) diagram to illustrate the regionalanatomy of the prefemoral area, and in particular the location ofthe coelomic aponeurosis, sartorius and iliacus muscles. Adaptedfrom Bojanus (1819).

some species, particularly aquatic chelonians (e.g.Loggerhead sea turtle, Caretta caretta), the post-pul-monary membrane is very thin and the lungs can be easilyvisualized. However, in many terrestrial chelonians (e.g.Greek tortoise, Testudo graeca) the post-pulmonary mem-brane, or septum horizontale, is more prominent making itimpossible to observe the lungs directly (Figure 25c,d).The heart lies outside the visceral coelomic cavity, withina distinct cranioventral pericardial sac, while the urinarybladder is variable in size and can occupy much of thedependent coelom (Figure 26a,b). Fluids and tissues canbe sampled using previously described techniques (Figure26c). Following insufflation gas removal, it is not neces-sary to repair the coelomic membrane or aponeurosisduring closure (Figure 26d). The skin is closed as previ-

ously described. Water-proofing the surgical site with tis-sue glue is recommended for aquatic species.

An extra-coelomic approach to the chelonian kidney hasalso been described (Hernandez-Divers, 2004). This tech-nique involves advancement of the sheathed telescope in acaudodorsal direction between the coelomic aponeurosisand the broad iliacus muscle. A combination of gentle lat-eral movements of the telescope tip coupled withintermittent insufflation is required to separate the coelom-ic aponeurosis from musculature, and reveal theretrocoelomic kidney(s).

Postoperative care — As long as insufflation gas is evac-uated, most reptiles recover quickly fromminimally-invasive endoscopy, but as with any surgicalprocedure continued provision of an appropriate thermalenvironment, analgesia, assisted ventilation, fluid therapy,and nutritional support should be considered.Antimicrobials are not routinely used following coe-lioscopy unless infection or contamination is identified atthe time of surgery.

DISCUSSION

Endoscopy is a surgical procedure and, as such, is limit-ed by any contraindication for general anesthesia, and theabilities of the surgeon. Debilitated animals should bemedically stabilized prior to coelioscopy. It is important to


Figure 20. Chelonian coelisocopic technique in a juvenile log-gerhead sea turtle (Caretta caretta). (A) Turtle positioned anddraped in lateral position with the telescope entry site marked(arrow); (B) following a 2 – 4 mm skin incision in the center ofthe fossa, the sheath and obturator are inserted and forced in acranial direction into the coelom; (C) the obturator is removedand replaced with the telescope, and the insufflation line isattached to the sheath (arrow); (D) examination can proceedusing a two-handed technique once the coelom is insufflated.The assistant is using a catheter via the operating channel toaspirate free fluid from the coelom.


Figure 21. Chelonian coelioscopy. (A) Normal liver in a Greektortoise (Testudo graeca) demonstrating multifocal pigmentatedareas of melanomacrophage aggregation; (B) diffusely pale liverin a male Hermann’s tortoise (Testudo hermanni) due to hepaticlipidosis; (C) pale area in the liver of a leopard tortoise(Geochelone pardalis) due to focal bacterial hepatitis; (D) liverbiopsy from a juvenile loggerhead sea turtle (Caretta caretta).

Figure 23. Chelonian coelioscopy. (A) immature ovary (arrow)in a loggerhead sea turtle (Caretta caretta); (B) ovary (1) largelyobscured by the closely associated infundibulum (2) and paleliver (3) in an adult female Greek tortoise (Testudo graeca), notethat increased hepatic fat is physiologic and normal during vitel-logenesis; (C) involuted oviduct in a juvenile leopard tortoise(Geochelone pardalis); (D) testis (1), vas deferens (arrow), epi-didmyis (2), and closely associated retrocoelomic kidney (3) in ared-eared slider (Trachemys scripta elegans).

Figure 24. Chelonian coelioscopy. (A) Retrocoelomic kidney(arrow) in a female Hermann’s tortoise (Testudo hermanni); (B)testis (1), retrocoelomic kidney (2) and vas deferens (3) in anadult male Greek tortoise (Testudo graeca); (C) incision throughthe coelomic membrane (1) using endoscopic scissors (2), toreveal the retrocoelomic kidney (3) of a box turtle (Terrapenecarolina); (D) biopsy from the retrocoelomic kidney of the samebox turtle.

Figure 22. Chelonian coelioscopy. (A) Stomach (1), liver (2) andcranial oviduct (3) in a Greek tortoise (Testudo graeca); (B)stomach (1), ileum (2), large intestine (3) and lung (4) in a log-gerhead sea turtle (Caretta caretta); (C) large intestine in a Greektortoise (Testudo graeca); (D) distended large intestine (arrow)due to impaction in a leopard tortoise (Geochelone pardalis).

remember that intracoelomic administration of fluids maysubsequently impede coelioscopy unless they are aspiratedat the beginning of the procedure. Stabilization is notalways possible and many procedures have been success-fully accomplished in moderate to high risk patients(Hernandez-Divers, 2004). Fluid therapy, assisted ventila-tion, thermal control, anesthetic monitoring, minimalsurgical trauma and species-specific anatomic knowledge,and reduced operating times compared to standard coe-liotomy are critically important for minimizing surgicalrisks.

Human endoscopists benefit from artificial teachingdevices and prolonged supervised instruction by experi-enced surgeons. Human laparoscopy trainers are expensiveand do not relate to the 2.7 mm system commonly used inreptile practice. In addition, there are limited opportunitiesto learn reptile endoscopy during traditional surgery orexotic animal residencies. Therefore, within the veterinaryfield initial instruction is best achieved through participa-tion in continuing education courses and practicallaboratories. While every opportunity should be taken topractice these techniques on cadavers, reptile carcassesrepresent a useful but imperfect model due to rapid deteri-oration after death. In those countries that permit andregulate the use of live animals for training veterinarians,non-recovery endoscopy laboratories using anesthetizedreptiles offer an unparalleled opportunity for establishingcompetence before embarking on clinical cases.

Endoscopy should ideally be performed after baselineclinicopathology and diagnostic imaging. The informationsuch diagnostic procedures provide assist in determiningstructures of primary interest, and the best endoscopicapproach. Entry of the 2.7 mm telescope, which is suitablefor the majority of reptiles presented to clinicians, onlyrequires a 2 – 4 mm skin incision and minimal blunt dis-section. The approaches described have not resulted in anysignificant morbidity. Insufflation is considered essentialfor reptile coelioscopy to provide sufficient telescope-tis-sue distance for examination and sample collection.Required insufflation pressures may varied from 0.4 – 0.7KPa (3 – 5 mmHg) for lizards, crocodilians, and cheloni-ans; however, pressures up to 1.4 Kpa (10 mmHg) wereoccasionally required in large snakes due to their reducedcoelomic space. Neverthessless, pressures were consistent-ly lower than those used in mammalian laparoscopy,typically 1.6 – 2.0 KPa (12 – 15 mmHg) (Magne andTams, 1999). Use of higher insufflation pressures in rep-tiles are likely to result in blood vessel compression andreduced venous return because of the lower diastolic bloodpressures of reptiles (Hicks, 1998).

Most reptiles lack any form of muscular diaphragm,although testudines, teiids, varanids, and crocodilians maypossess post-pulmonary and/or post-hepatic membranesthat may be substantial (Perry, 1998). The lack of a true,muscular diaphragm does result in lung compression dur-ing coelomic insufflation even at the relatively low


Figure 25. Chelonian coelioscopy. (A) Spleen (1), stomach (2)and liver (3) in a Herman’s tortoise (Testudo hermanni); (B) pan-creas (1) adjacent to the duodenum (2) in a box turtle (Terrapenecarolina); (C) lung (1), stomach (2) and liver (3) in a loggerheadsea turtle (Caretta caretta), note the absence of a major post-pul-monary membrane between the telescope and the lung; (D)post-pulmonary membrane (1) in a Greek tortoise (Testudo grae-ca) that obscures direct visualization of the lungs, liver (2) alsovisible.

Figure 26. Chelonian coelioscopy. (A) Heart (1) within a sepa-rate pericardium, and cranial border of the liver (2) in a red-earedslider (Trachemys scripta elegans); (B) urinary bladder (1) andcaudal liver (2) in a African spurred tortoise (Geochelonepardalis); (C) aspiration of free coelomic fluid using an injec-tion/aspiration needle (1) close to the liver (2) and small intestine(3) in a loggerhead sea turtle (Caretta caretta); (D) coelioscopicentry site showing the perforated coelomic membrane (1) andcoelomic aponeurosis (2).

pressures recommended. Tidal volume is maintained whenusing a volume-cycle ventilator but with pressure-cycleventilators the inspiration pressure should be increased tocounteract the effects of insufflation on pulmonary func-tion. Most procedures are performed with the animal inlateral or dorsal recumbency and although no adverseeffects have been noted as a consequence, it is possiblethat the dependent lung may be collapsed by the weight ofoverlying viscera. Postural factors should be considered aspotential causes of ventilation-perfusion mismatches, par-ticularly in large reptiles (Wang, et al, 1998).

Carbon dioxide is recommended for insufflation inhuman and veterinary laparoscopy because it is readilyabsorbed, rapidly eliminated, and has been associated withfewer complications (Golditch, 1971, Magne and Tams,1999). However, on the rare occasions when air was used,no deleterious effects were observed ((Divers, 1998,2000). In all cases pneumocoelom should be resolved priorto closure to reduce post-operative discomfort.

Magnification provided by the telescope assists with theidentification and biopsy of lesions, with minimal collater-al damage to adjacent structures. Instruments used throughthe operating channel of the sheath enables visualizationand biopsy through a single-entry technique in most rep-tiles under 10 kg. In larger animals, two or three ports(telescope and one or two instruments) can be triangulated,but again a surgeon can usually accomplish diagnostic pro-cedures unassisted.

There are very few pathognomonic signs or consistentclinicopathologic changes associated with known diseasestates of reptiles. In many cases a definitive diagnosis ofdisease relies upon the demonstration of a host pathologicresponse, and if infectious, culture and identification of thecausative pathogen. Tissue biopsies are, therefore, fre-quently essential, and endoscopic biopsy provides aminimally-invasive technique for their collection. Correcthandling of biopsy forceps and collected tissue reducescrush artifact, improves biopsy quality, and enhanceshistopathologic interpretation. For example, the results of arenal biopsy study in 23 green iguanas indicated that endo-scopic biopsy collection produced excellent samples withnegligible trauma to the patient (Hernandez-Divers, et al,2004b). Common alternatives to endoscopic biopsyinclude conventional surgical and ultrasound-guided tech-niques. Reports from human surgeons indicate thatconsiderable benefits may be gained from minimally-inva-sive endoscopic surgery, compared to other techniques(Golditch, 1971, Corson and Grochmal, 1990, VanderVelpen, et al, 1994, Yu, et al, 1997, Kehlet, 1999, Lagares-Garcia, et al, 2003). Human laparoscopy has been creditedwith more rapid and accurate diagnosis, reduced need forextensive laparotomy, reduced surgical stress, improvedpostoperative pulmonary function, reduced hypoxemia,

reduced surgical time, and faster recovery (Yu, et al, 1997,Kehlet, 1999). The disadvantage of human laparoscopyappears minimal and restricted to misdiagnosis in less than1% of cases. No significant morbidity has been demon-strated with appropriate laparoscopic technique (VanderVelpen, et al, 1994). In the few comparative studies havebeen published in veterinary medicine, endoscopic tech-niques provided superior sample quality with reducedcomplication rates compared to ultrasound-guided proce-dures (Kovak, et al, 2002, Rawlings, et al, 2003).

The most substantial limitation to successful ante-mortem diagnosis is the relative small size and delicatenature of most reptiles. Both of these limitations can belargely overcome using diagnostic endoscopy which pro-vides focal magnification, illumination, andminimally-invasive surgical access to the coelom. Obesityis a frequent hindrance in mammals, but the lack of exten-sive fat deposition around the visceral organs of mostreptiles (except for the diffuse fat bodies of snakes) makesthis less of a concern. However, inappropriate patient posi-tioning or telescope entry into a fat body will certainlyhinder endoscopic evaluation. Large bladders, voluminousintestinal tracts and active female reproductive systemscan present more serious obstacles that should be appreci-ated and avoided. In addition, order, suborder and familydifferences in anatomy necessitate the application of gen-eral principles rather than rigidly adhered to techniques.For example, the ability to perform prefemoral coe-lioscopy in a chelonian is affected by the shape andconformation of the prefemoral fossa and shell.

No significant morbidity has been demonstrated withappropriate laparoscopic technique in humans (VanderVelpen, et al, 1994). The efficacy, complications, and longterm effects of coelioscopy have not been extensively doc-umented in reptiles, although previous and on-goingstudies at the University of Georgia continue to criticallyevaluate these procedures (Hernandez-Divers, 2004,Hernandez-Divers, et al, 2004a, Hernandez-Divers, et al,2004b).

ACKNOWLEDGEMENTS

The authors thanks Karl Storz Veterinary Endoscopy forproviding photographs for figures 1B and 1C, and for theircontinued support of the endoscopy training, research, anddevelopment at the College of Veterinary Medicine,University of Georgia. We also thank our colleagues thatassisted with endoscopic procedures used in writing thisreview, including Drs. Clarence Rawlings, HeatherWilson, Anneliese Strunk, Christopher Hanley, andMichael McBride. Thanks also to Kip Carter ofEducational Resources for preparing the illustrations usedin figures 5B and 6.


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JAVMA, Vol 233, No. 3, August 1, 2008 Scientific Reports 1

SM

ALL A

NIM

ALS

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XO

TIC

The ball python (Python regius) is a medium-sized snake of the family Boidae and is native to West

Africa. Because of its gentle nature, moderate size, and variably attractive skin patterns, this snake is a popular species maintained in captivity. The respira-tory system of snakes has been extensively reviewed.1 The trachea is long and narrow and composed of in-complete cartilaginous rings that are supported by a dorsal ligament. The trachea terminates into 2 short primary bronchi because ball pythons, like other boids, have both left and right lungs. Each bronchus continues a short distance as an intrapulmonary bronchus before terminating in the cranial portion of the lung. Each lung is composed of 3 areas: a highly

Evaluation of transcutaneous pulmonoscopy for examination and biopsy of the lungs

of ball pythons and determination of preferred biopsy specimen handling and fixation procedures

Scott J. Stahl, dvm, dabvp; Stephen J. Hernandez-Divers, bvetmed, dzoomed, daczm; Tanya L. Cooper; Uriel Blas-Machado, dvm, phd, dacvp

JAVMA—07-11-0582—Stahl—2 Fig—2 Tab—NJR—HLS

Objective—To establish a safe and effective technique for the endoscopic examination and biopsy of snake lungs by use of a 2.7-mm rigid endoscope system.Design—Prospective study.Animals—17 subadult and adult ball pythons (Python regius).Procedures—The right lung of each anesthetized snake was transcutaneously penetrated at a predetermined site. Endoscopic lung examination was objectively scored, and 3 lung biopsies were performed. Tissue samples were evaluated histologically for diagnostic qual-ity. One year later, 11 of the 17 snakes again underwent pulmonoscopy and biopsy; speci-mens were placed in various fixatives to compare preservation quality. All 17 snakes were euthanatized and necropsied.Results—No major anesthetic, surgical, or biopsy-associated complications were detected in any snake. In 16 of 17 pythons, ease of right lung entry was satisfactory to excellent, and views of the distal portion of the trachea; primary bronchus; intrapulmonary bronchus; cranial lung lobe; and faveolar, semisaccular, and saccular lung regions were considered excellent. In 1 snake, mild hemorrhage caused minor procedural difficulties. After 1 year, pulmonoscopy revealed healing of the previous transcutaneous lung entry and biopsy sites. Important procedure-induced abnormalities were not detected at necropsy. Diagnostic quality of specimens that were shaken from biopsy forceps into physiologic saline (0.9% NaCl) solution before fixation in 2% glutaraldehyde or neutral-buffered 10% formalin was considered good to excellent.Conclusions and Clinical Relevance—By use of a 2.7-mm rigid endoscope, lung examination and biopsy can be performed safely, swiftly, and with ease in ball pythons. Biopsy specimens obtained during this procedure are suitable for histologic examination. (J Am Vet Med Assoc 2008;233:xxx–xxx)

vascular faveolar region in which gaseous exchange occurs; a short semisaccular (transitional) zone; and a larger saccular area, which is thin, semitransparent, and poorly vascularized.

As in other species of captive snakes, bacterial and fungal respiratory diseases are common in ball pythons and are often related to suboptimal tem-perature, humidity, or ventilation.2 In addition, para-myxovirus-associated respiratory tract disease in boids and tracheal chondromas in ball pythons have been reported.3 Given that specific treatment requires accurate diagnosis, the collection of exudates and tis-sue samples from the respiratory tract is important.4 Although various sampling techniques have been de-scribed, endoscopy provides the least invasive means of direct lung examination and biopsy and has been described for snakes and other reptiles.5–8 The long narrow trachea of snakes makes it difficult to impos-sible to use most rigid and flexible endoscopes to evaluate the distal portion of the trachea and lung via an endotracheal approach. Consequently, trans-cutaneous insertion of a rigid endoscope directly into the lung has been advocated.5,7 The purpose of the

From Stahl Exotic Animal Veterinary Services, 111A Center St S, Vienna, VA 22180 (Stahl); and the Department of Small Animal Medicine and Surgery (Hernández-Divers, Cooper) and Athens Vet-erinary Diagnostic Laboratory (Blas-Machado), College of Veteri-nary Medicine, University of Georgia, Athens, GA 30602-7390.

The authors thank Lisa Holthaus and Jason Norman for technical as-sistance and Karl Storz Veterinary Endoscopy America Inc and BAS Vetronics-Bioanalytical Systems Inc for provision of equipment.

Address correspondence to Dr. Hernandez-Divers.

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study reported here was to establish a safe and effec-tive technique for transcutaneous endoscopic exami-nation and biopsy of the lungs of snakes by use of a 2.7-mm rigid endoscope.

Materials and Methods

Animals—Seventeen recently imported adult ball pythons (15 females and 2 males) were obtained from a reptile wholesaler for use in the study. All pro-cedures and methods were reviewed and accepted by the University of Georgia’s Institutional Animal Care and Use Committee (IACUC No. A2006-10076-0). The pythons were maintained in conditions approved by the Association for Assessment and Accreditation of Laboratory Animal Care. Snakes were housed in groups of 3 or 4 in large plastic containers main-tained in a room at an ambient temperature of 24°C (75°F) during the night and 27°C (81°F) during the day. Mercury halide incandescent lamps that were suspended above each enclosure provided a daytime basking area at 35°C (95°F). Pythons were exposed to a repeating cycle of 12 hours of light followed by 12 hours of darkness and a general humidity level of 50%. The snakes were physically examined on ar-rival and found to be clinically normal adults. The snakes were acclimatized to the research facilities for 7 days prior to the start of the study. They were not offered food during this acclimatization period, but water was available at all times. Following the sur-gical procedures, snakes were offered frozen-thawed rodents weekly.

On the day of surgery, the ball pythons were trans-ferred to heated incubators at 29°C (85°F) for at least 1 hour prior to commencement of experimental proce-dures. The examination, anesthesia, and surgery areas were maintained at 24°C. Body weight and resting re-spiratory and heart rates were recorded for each snake. Each snake was identified by use of a unique number written with a permanent marker pen on the dorsal as-pect of the cranium.

Anesthesia—Each python was premedicated with butorphanol tartratea (1 mg/kg [0.45 mg/lb]) adminis-tered via injection into the epaxial muscles 20 minutes prior to induction of anesthesia via intracardiac injec-tion with propofolb (5 mg/kg [2.27 mg/lb]). Following intubation, anesthesia was maintained by use of 1% to 3% isoflurane in 100% oxygen (flow rate, 1 L/min) and adjusted to the individual’s requirements. Throughout the anesthetic period, assisted ventilation was provid-ed by use of a pressure-cycle ventilatorc; adjustments were made to maintain end-tidal CO2 readings > 10 mm Hg. Hypothermia was minimized by placing the snake on recirculating warm water blanketsd that were set to 40°C (105°F). Monitoring included assessments of tongue and tail withdrawal reflexes and ventral muscle tone, end-tidal capnography,e cardiac Doppler ultrasonography,f pulse oximetry,g and esophageal tem-perature measurement.h

Endoscopy—Each python was positioned in left lateral recumbency (with the dorsum facing the surgeon) on a horizontally level surgery table. The surgical entry site was identified at 90 ventral scales

caudal to the head and 9 scales lateral on the right side. Following aseptic preparation, a vertical 8- to 10-mm incision was made through the interscalar skin. The subcutis was bluntly dissected until the underlying ribs and intercostal space were identified. Small straight mosquito hemostats were used to pen-etrate the intercostal muscles and separate the 2 ad-jacent ribs. The serosal surface of the right lung was identified as a thin semitransparent membrane con-taining a latticework of small blood vessels, which inflated in association with ventilation. The lung was penetrated by use of small hemostats to create a 3- to 4-mm pneumotomy and facilitate insertion of the 30° telescope (2.7 mm X 18 cm) that was housed within a 14.5-F operating sheath and connected to a xenon light source, endovideo camera, monitor, and digital recorder.i

Endoscopic examinations were performed by 2 experienced reptile endoscopists (SJS and SJHD). Each endoscopist scored the ease of entry into the lung (including skin incision, hemostat penetration, and entry of the endoscope) on a scale from 1 to 5 (1 = impossible [interval to insertion of endoscope, > 15 minutes]; 2 = difficult [interval to insertion of endoscope, 11 to 15 minutes]; 3 = satisfactory [in-terval to insertion of endoscope, 6 to 10 minutes]; 4 = good [interval to insertion of endoscope, 2 to 5 minutes]; and 5 = excellent [interval to insertion of endoscope, < 2 minutes]). Additionally, the endos-copist scored the ease of location and observation of various structures associated with the right side of the lower respiratory tract, including the distal por-tion of the trachea; primary bronchus; intrapulmo-nary bronchus; and regions of faveolar (cranial, vas-cular) lung, semisaccular (transitional zone) lung, and saccular (avascular air sac) lung on a scale of 1 to 5 (1 = impossible, 2 = difficult, 3 = satisfactory, 4 = good, and 5 = excellent).

Biopsy specimen collection—Once the evaluation was completed, 3 biopsies were performed endoscopi-cally; samples were collected from the right faveolar region by use of 5-F biopsy forcepsi through the instru-ment channel of the endoscope sheath. Each biopsy specimen was gently transferred from the forceps to a biopsy cassette by use of a moistened cotton-tipped applicator; the cassette was then closed and placed in neutral-buffered 10% formalin. Hemorrhage from the biopsy sites was recorded on a scale of 1 to 3 (1 = no hemorrhage, 2 = minor hemorrhage, and 3 = major hemorrhage).

Completion of procedure—Only the skin was closed by use of a single 4-0 polydioxanonej horizon-tal mattress suture. Any complications associated with the anesthetic or surgical procedures were recorded. Eleven snakes were permitted to recover from anes-thesia and were provided with postoperative analgesia (0.2 mg of meloxicamk/kg [0.09 mg/lb], IM). Six py-thons were not permitted to recover but were euthana-tized via IV injection of pentobarbital for immediate necropsy.

Repeat pulmonoscopy and biopsy specimen col-lection—The 11 remaining snakes were maintained


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for 12 months before undergoing repeat anesthesia and transcutaneous pulmonoscopy, as described. This sec-ond procedure was not scored, and the entry site was located at 95 ventral scales caudal to the head and 9 scales lateral on the right side to facilitate examination of the previous surgical approach. The right lung was evaluated for signs of disease or trauma that could be associated with the previous surgery. In particular, the surgical entry site into the lung and biopsy sites were evaluated.

Three endoscopic biopsy specimens were collected from the faveolar region of each snake (33 in total), but to avoid any physical damage to the harvested tissue, each biopsy was gently shaken from the forceps into a sterile red-top blood collection tube containing 1 mL of physiologic sterile saline (0.9% NaCl) solution. The sterile saline solution was then decanted and replaced with 1 of 3 fixatives; neutral-buffered 10% formalin so-lution, 2% glutaraldehyde, or Davidson’s medium. Bi-opsy specimens were processed routinely for histologic evaluation.

Necropsy and histologic examination of tis-sue—Six pythons were euthanatized via IV admin-istration of pentobarbital immediately following the original endoscopic procedure, and each snake un-derwent a full gross necropsy examination. The re-maining 11 snakes were similarly euthanatized and examined 12 months later, immediately following the second endoscopy procedure. In all instances, the right lung was evaluated for any evidence of trauma or disease, and samples of lung (and any other abnormal tissues) were collected into neutral-buffered 10% formalin for routine histologic evalu-ation. Biopsy and necropsy tissues were processed routinely, embedded in paraffin, sectioned at ap-proximately 5 µm, stained with H&E stain, and ex-amined microscopically. Histologically, biopsy and necropsy tissues were subjectively compared to de-termine whether biopsy specimens collected during endoscopy were representative of tissue collected during necropsy. In addition, the diagnostic quality of each biopsy specimen was scored on a scale of 1 to 4 (1 = nondiagnostic, 2 = poor, 3 = good, and 4 = excellent); the criteria used included relative size of the biopsy sample in relation to the area biopsied, presence of crushing artifacts, quality of architec-tural detail preservation achieved via fixation, and tinctorial quality of the stained tissue.

Results

Among the 17 ball pythons, mean ± SD body weight and snout-to-vent length were 1,348 ± 327 g (2.972 ± 0.721 lb) and 109.9 ± 9.4 cm, respectively. All snakes appeared to be clinically normal adults and were in acceptable body condition. There were no sig-nificant changes in body weights during the course of the study. Premedication with butorphanol, induc-tion of anesthesia with propofol, and maintenance of anesthesia with isoflurane in oxygen via intermittent pressure ventilation resulted in a surgical plane of an-esthesia without complications in all snakes. Pre- and intraoperative variables were recorded (Table 1).

Endoscopy score data were not normally distrib-uted (Table 2). All mean endoscopy scores were > 4 (good), and the mean hemorrhage score was only 1.1 (Figure 1). In 16 of 17 snakes, the ease of entry score was considered satisfactory to excellent; however, in 1 snake, entry was considered difficult (score of 2) because of mild hemorrhage following hemostat pen-etration into the lung. Observation of the structures associated with the lower respiratory tract (accessed via the right lung), including the distal portion of the trachea; primary bronchus; intrapulmonary bron-chus; cranial lung lobe; and faveolar, semisaccular, and saccular lung regions, was considered excellent (score of 5) in 16 of 17 pythons. In the snake with mild hemorrhage, observations of the trachea and primary bronchus were considered good (score of 4) and satisfactory (score of 3), respectively. However, endoscopic examination and biopsy procedures were still performed without complication in that snake.

Variable MeanSD

Preoperative respiratory rate (breaths/min) 8.3 4.9Preoperative heart rate (beats/min) 44 8Butorphanol premedication dose* (mg/kg) 1.0 0.03Propofol dose† for induction of anesthesia (mg/kg) 5.2 1.4

Intraoperative ventilation rate‡ (breaths/min) 6.4 0.9Intraoperative maximum inspiratory pressure (mm Hg) 4.0 0.9Intraoperative end-tidal CO2 pressure (mm Hg) 12.4 1.7Intraoperative heart rate (beats/min) 32 7Intraoperative esophageal temperature (°C[°F]) 27.4 0.8 (81.3 1.4)

*Administered via injection into the epaxial muscles. †Adminis-tered via intracardiac injection. ‡Assisted ventilation was provided throughout the anesthetic period by use of a pressure-cycle ven-tilator.

To convert kilograms to pounds, multiply by 2.2.

Table 1—Pre- and perioperative findings in 17 ball pythons that un-derwent transcutaneous rigid pulmonoscopy of the right lung.

Variable Score

Ease of initial entry* 4.2 1.0Ease of location and observation of various structures† Distal portion of the trachea 4.9 0.2 Primary bronchus 4.9 0.5 Intrapulmonary bronchus 5.0 0.0 Faveolar lung region 5.0 0.0 Semisaccular lung region 5.0 0.0 Saccular lung region 5.0 0.0Postbiopsy hemorrhage‡ 1.1 0.3

*Ease of entry into the lung was assessed on a scale from 1 to 5 (1 = impossible [interval to insertion of endoscope, 15 min]; 2 = difficult [interval to insertion of endoscope, 11 to 15 min]; 3 = satis-factory [interval to insertion of endoscope, 6 to 10 min]; 4 = good [interval to insertion of endoscope, 2 to 5 min]; and 5 = excellent [interval to insertion of endoscope, 2 min]). †Ease of location and observation of various structures was assessed on a scale of 1 to 5 (1 = impossible, 2 = difficult, 3 = satisfactory, 4 = good, and 5 = excellent). ‡Hemorrhage from the biopsy sites was assessed on a scale of 1 to 3 (1 = no hemorrhage, 2 = minor hemorrhage, and 3 = major hemorrhage).

Table 2—Assessments (mean ± SD scores) of ease of entry into the right lung, ease of observation of anatomic structures, and hemorrhage from biopsy sites in 17 ball pythons that were anes-thetized and underwent transcutaneous rigid pulmonoscopy of the right lung.

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Among the 17 snakes, minor hemorrhage was rare-ly associated with entry into the lung but more com-monly developed following lung biopsy. In the initial experiment, biopsy procedures were associated with minor and clinically unimportant bleeding in 15 of 17 snakes. In 2 snakes, intraoperative bleeding was con-sidered severe in the endoscopic views, but in one of those snakes, no major hemorrhage was identified dur-ing necropsy performed immediately after completion of the experiment. The other affected python recovered

completely and uneventfully, without any evidence of mucous membrane pallor asso-ciated with severe hemorrhage.

All 11 snakes that were allowed to re-cover from aesthesia did so uneventfully, without any evidence of morbidity and with no deaths associated with the proce-dure. Repeat pulmonoscopy 1 year later re-vealed healing of the previous biopsy sites, which appeared as small defects within the faveolar lung; typically, the original pneu-motomy site was barely visible as a small scar.

Necropsy examinations did not reveal any notable damage to the skin, subcutis, and pulmonary or other visceral tissues. Diagnostic quality scores of the biopsy specimens obtained via pulmonoscopy were assessed; criteria for score alloca-tion included the presence of crushing artifacts, preservation of architectural detail, and tinctorial quality of H&E-stained tissues. Specimens collected from 17 snakes (3 specimens/snake; 51 biopsy specimens in total) during the initial en-doscopic procedure were transferred to a biopsy cassette by use of a cotton-tipped applicator, and the cassette was placed in neutral-buffered 10% formalin; for these samples, mean ± SD quality score was 2.5 ± 0.8. Specimens were also collected from 11 of those snakes during a repeat proce-dure 1 year later (3 specimens/snake; 33 biopsy specimens in total). Samples were each shaken from forceps into saline solu-tion and transferred to a biopsy cassette prior to placement in neutral-buffered 10% formalin, glutaraldehyde, or David-son’s medium; for these samples, mean ± SD quality score was 3.0 ± 0.0, 3.3 ± 0.5, and 2.0 ± 0.0, respectively.

Regardless of the fixative solution and method used for tissue handling, biopsy specimens were well-fixed and representative of the luminal half of the faveolar lung (Figure 2). Although most of the specimens were of adequate size, some were too small or were crushed during collection or transfer to the fixa-tives. In other specimens, the faveolar septae were clumped or collapsed; such artifact was detected most frequently in specimens that were transferred from

forceps directly to fixative solution by use of cot-ton-tipped applicators. Architectural integrity and detail of the biopsy specimens were well preserved in all fixatives, but the tinctorial quality was best maintained in samples placed in the 2% glutaral-dehyde solution; tinctorial quality was somewhat less well maintained in samples placed in neutral-buffered 10% formalin. Preservation of the tincto-rial quality was inadequate in tissues fixed with Davidson’s medium.

Figure 1—Representative endoscopic views obtained from 17 ball pythons that were anesthetized and underwent transcutaneous rigid endoscopy of the right lung. A—Cranial view of the faveolar lung region. B—Close-up view of the faveo-lar lung region to illustrate the primary (p), secondary (s), and tertiary (t) septae of the vascular portion of the lung. C—View of the semisaccular (or transitional zone) region of the lung. D—Caudal view of the saccular lung region illustrating its thin nature and poor vascularity. E—View of the cranial aspect of the faveolar lung region illustrating the intrapulmonary bronchus (i), short primary bronchus (b), trachea (t), and anterior lung lobe (a). F—Close-up view illustrating the intra-pulmonary bronchus (i), short primary bronchus (b), trachea (t), and anterior lung lobe (a). G—View into the anterior lung lobe (a) in which the primary bronchus (b) is also visible. H—View inside the distal portion of the trachea (t). Notice the incomplete cartilaginous rings and dorsal ligament (d). I—View of a biopsy pro-cedure in the faveolar lung region involving use of 5-F biopsy forceps. J—View to illustrate the minimal bleeding that was typically observed immediately following a biopsy procedure. K—View of a typical healed biopsy site after an interval of 1 year. Notice the healed defect within the faveolar tissue. b = Primary bronchus. L—View of an original pneumotomy entry site after an interval of 1 year. Notice the complete healing and minimal scarring (arrow) of the tissues.


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Discussion

The use of ball pythons for evaluation of the safety and effectiveness of rigid endoscopy to examine the lower respiratory tract of snakes was highly successful. The right lung was chosen for this procedure because the left lung is either absent or reduced in most snakes.1 However, in boids with disease that affects the left lung, a left (or bilateral) approach could be undertaken. The endoscope entry site into the right lung was specifically determined to coincide with the reduced vascularity of the semisaccular (transitional) portion of the lung. In addition to minimizing hemorrhage, entry at this level permitted an excellent view as far cranial as the distal portion of the trachea and as far caudal as the caudal extent of the saccular lung (air sac). In ball pythons, the entry site was located at 90 ventral scales caudal to the head and 9 scales lateral on the right side. This equates to 44% of the total snout-to-vent length. This landmark was determined by the anatomic evaluation and scale counts of several dissected specimens and published morphometric data.1 Jekl and Knotek7 suggest a simi-lar entry point (35% to 45% of the total snout-to-vent length) for ball pythons, boa constrictors (Boa constric-tors), and Burmese pythons (Python molurus bivittatus).

Extensive morphometric data for many species of snakes has been summarized1 and can be used to accurately determine en-try into the semisaccular lung. However, in the authors’ experience, entry into the right lung of most snake species can be approxi-mated by identifying the location of the heart and selecting a point halfway between the heart and the vent (ie, at approx 40% to 45% of the total snout-to-vent length).

Propofol and isoflurane provided ef-fective and controllable anesthesia in the ball pythons of the present study. End-tidal CO

2 values have been poorly investigated

in reptiles. Observations in green iguanas have indicated that there may be poor cor-relation between end-tidal CO

2 and arte-

rial PCO2 values because of intracardiac or

intrapulmonary shunting.9 However, clini-cal observations by the authors have sug-gested that maintaining the end-tidal CO

2

value at > 10 mm Hg in snakes reduces the time to return to unassisted respiration fol-lowing anesthesia. Insertion and movement of the endoscope within the right lungs of the study snakes did not appear to interfere with the maintenance of surgical anesthesia and did not alter the measured physiologic variables; however, the ability to accurately control and maintain respiration by use of the electrical ventilator was likely essential. A tight seal between the endoscope and the snake’s skin, combined with closure of all the sheath ports, was important for prevent-ing gas exchange across the surgical site. If a port were accidentally left open, the pressure cycle ventilator would not trigger and end-tidal CO

2 values would decrease to zero.

The surgical approach used for endoscopic lung examination in snakes in the present study was more lateral and less extensive but otherwise similar to that described previously.7 In 16 of the 17 snakes, the ease of entry score was considered satisfactory to excellent; however, in 1 snake, entry was considered somewhat difficult because of mild hemorrhage following hemo-stat penetration into the lung. Endoscopic evaluation of the structures associated with the right lower respirato-ry tract was considered excellent in 16 of 17 pythons; in the snake with hemorrhage, observations of the trachea and primary bronchus were considered good and sat-isfactory, respectively, but this did not impede comple-tion of the examination and biopsy procedures.

Minor hemorrhage was occasionally associated with entry into the lung but most commonly evident following lung biopsy. Biopsy procedures were associ-ated with minor bleeding in 15 of the 17 snakes. In 2 snakes, intraoperative bleeding appeared severe endo-scopically; in 1 snake, no major hemorrhage was de-tected at necropsy immediately after the endoscopic examination, and the other recovered without clinical signs of severe hemorrhage. Although pre- and postop-erative Hct values were not determined, on the basis of the clinical and necropsy findings, we concluded

Figure 2—Photomicrographs of sections of biopsy specimens collected during endoscopy from the faveolar lung region of ball pythons. A—Representative low-magnification image of a section of lung tissue that was gently shaken into physi-ologic saline (0.9% NaCl) solution before fixation in 2% glutaraldehyde. Notice that there are minimal crushing artifacts, and the preservation of architectural de-tail by fixation and tinctorial quality of this tissue are excellent. H&E stain; bar = 318 µm. B—Representative high-magnification image of a section of a lung tissue specimen that was gently shaken into saline solution before fixation in Davidson’s medium. Preservation of architectural detail by fixation and tinctorial quality of this tissue are poor. H&E stain; bar = 35 µm. C—Representative high-magnification image of a section of a lung tissue specimen that was gently shaken into saline solution before fixation in neutral-buffered 10% formalin. Notice that there are minimal crushing artifacts, and the preservation of architectural detail by fixation and tinctorial quality of this tissue are excellent. H&E stain; bar = 35 µm. D— Rep-resentative high-magnification image of a section of a lung tissue specimen that was gently shaken into saline solution before fixation in 2% glutaraldehyde. No-tice that there are minimal crushing artifacts, and the preservation of architectural detail from fixation and tinctorial quality of this tissue are excellent. H&E stain; bar = 35 µm.

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that hemorrhage was minor and clinically unimportant but was magnified by the optics of the endoscope. All snakes permitted to recover from anesthesia after the initial evaluation did so uneventfully and without any evidence of morbidity and with no deaths during the following 12-month period.

Lung biopsy tissues are delicate. In the present study, lung tissue architecture was damaged during transfer of specimens by use of cotton-tipped applica-tors, resulting in poor to satisfactory diagnostic quality (mean quality score, 2.5). Diagnostic quality of tissues was improved by gently shaking specimens free from the forceps into sterile saline solution, before proceed-ing with fixation in neutral-buffered 10% formalin or glutaraldehyde (mean quality score, 3.0 and 3.3, respec-tively). Compared with those fixation techniques, use of Davidson’s medium resulted in poorer staining (mean quality score, 2.0) and is therefore not recommended for processing of snake lung biopsy specimens.

Transcutaneous pulmonoscopy appears to be safe and effective for examination of the lower respiratory tract of snakes and is recommended when fine-diam-eter flexible or rigid endoscopes cannot reach the lungs via an endotracheal approach. Furthermore, biopsy procedures performed during lung endoscopy appear to be tolerated well and yield tissue samples of diag-nostic quality.

a. Torbugesic (10 mg/mL), Fort Dodge Animal Health, Overland Park, Kan.

b. Propofol (10 mg/mL), Abbott Laboratories, North Chicago, Ill.c. VT-5000, BAS Vetronics, Bioanalytical Systems Inc, West Lafay-

ette, Ind.d. Temperature therapy pad TP22G and temperature therapy

pump TP500T, Gaymar, Orchard Park, NY.

e. ETCO2/SpO

2 monitor, CO

2 SMO, Novametrix Medical Systems,

Wallingford, Conn.f. Ultrasonic Doppler, Parks Electric Laboratory, Aloha, Ore.g. V3301 Pulse Oximetry, SurgiVet, Waukesha, Wis.h. Precision Thermometer, Tandy, Fort Worth, Tex.i. 64018BSA, 67065C, 201320-20, 69235106, 9291-B, 20094002U,

and 67161 Z, Karl Storz Veterinary Endoscopy America Inc, Go-leta, Calif.

j. PDS II, 2 metric, Ethicon, Somerville, NJ.k. Metacam (5 mg/mL, injectable), Boehringer Ingelheim Vetmed-

ica Inc, St Joseph, Mo.

References1. Wallach V. The lungs of snakes. In: Gans C, Gaunt AS, eds. Biology

of the reptilia. Vol 19. Ithaca, NY: Society for the Study of Amphib-ians and Reptiles, 1998;93–295.

2. Murray MJ. Pneumonia and lower respiratory tract disease. In: Mader DM, ed. Reptile medicine and surgery. 2nd ed. St Louis: Elsevier, 2006;865–877.

3. Drew ML, Phalen DN, Berridge BR, et al. Partial tracheal ob-struction due to chondromas in ball pythons (Python regius). J Zoo Wildl Med 1999;30:151–157.

4. Hernandez-Divers SJ. Diagnostic techniques. In: Mader DR, ed. Reptile medicine and surgery. 2nd ed. St Louis: Elsevier, 2006; 490–532.

5. Hernandez-Divers SJ. Diagnostic and surgical endoscopy. In: Raiti P, Girling S, eds. Manual of reptiles. 2nd ed. Cheltenham, England: British Small Animal Veterinary Association, 2004; 103–114.

6. Hernandez-Divers SJ, Shearer D. Pulmonary mycobacteriosis caused by Mycobacterium haemophilum and M. marinum in a royal python. J Am Vet Med Assoc 2002;220:1661–1663.

7. Jekl V, Knotek Z. Endoscopic examination of snakes by access through an air sac. Vet Rec 2006;158:407–410.

8. Taylor WM. Endoscopy. In: Mader DR, ed. Reptile medicine and surgery. 2nd ed. St Louis: Elsevier, 2006;549–563.

9. Schumacher J, Yelen T. Anesthesia and analgesia. In: Mader DR, ed. Reptile medicine and surgery. 2nd ed. St Louis: Elsevier, 2006;442–452.

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JAVMA, Vol 230, No. 12, June 15, 2007 Scientific Reports: Original Study 1849

In small animal medicine, various antemortem tech-niques for the collection of liver tissue samples for

diagnostic purposes have been tried and tested. Cur-rently, percutaneous, endoscopic, or open surgical pro-cedures are most commonly used to collect samples for cytologic or histologic examination.1-3 Percutane-ous biopsy procedures allow liver tissue samples to be safely collected by use of biopsy needles in dogs that weigh > 10 kg (22 lb); fine-needle aspiration by use of smaller gauge hypodermic needles is preferred in smaller animals.1,2 In most instances, collection of such samples is performed with ultrasound guidance. Comparisons of liver biopsy specimens and fine-needle aspiration samples obtained from dogs and cats have revealed that findings of cytologic examination of aspi-rates is only diagnostic in 30% to 61% of samples, com-pared with findings of histologic examination of tissue

Evaluation of an endoscopic liver biopsy technique in green iguanas

Stephen J. Hernandez-Divers, bvetmed, dzoomed, daczm; Scott J. Stahl, dvm, dabvp; Michael McBride, dvm; Nancy L. Stedman, dvm, phd, dacvp

From the Departments of Small Animal Medicine and Surgery (Hernandez-Divers, McBride) and Pathology (Stedman), College of Veterinary Medicine, University of Georgia, Athens, GA 30602; and Stahl Exotic Animal Veterinary Services, 111A Center St S, Vienna, VA 22180 (Stahl). Dr. McBride’s present address is Roger Williams Park Zoo, 1000 Elmwood Ave, Providence, RI 02907. Dr. Stedman’s present address is Wyeth Research, 1 Burtt Rd, Andover, MA 01810.

The authors thank Karl Storz Veterinary Endoscopy America Inc and BAS Vetronics-Bioanalytical Systems Inc for provision of research equipment.

Address correspondence to Dr. Hernandez-Divers.

Objective—To establish a safe and effective endoscopic technique for collection of liver bi-opsy specimens from lizards by use of a 2.7-mm rigid endoscope system that is commonly available in zoologic veterinary practice.Design—Prospective study.Animals—11 subadult male green iguanas (Iguana iguana).Procedures—Each lizard was anesthetized, and right-sided coelioscopic examination of the right liver lobe and gallbladder was performed. Three liver biopsy specimens were collected from each lizard by use of a 2.7-mm rigid endoscope and 1.7-mm (5-F) biopsy forceps. Bi-opsy samples were evaluated histologically for quality and crush artifact. Ten days following surgery, all iguanas were euthanatized and underwent full necropsy examination. Results—For all 11 iguanas, the right liver lobe and gallbladder were successfully examined endoscopically, and 3 biopsy specimens of the liver were collected without complications. Mean ± SD durations of anesthesia and surgery were 24 ± 7 minutes and 6.8 ± 1.0 minutes, respectively. At necropsy, there was no evidence of trauma or disease associated with the skin or muscle entry sites, liver, or any visceral structures in any iguana. All 33 biopsy speci-mens were considered acceptable for histologic interpretation; in most samples, the extent of crush artifact was considered minimal.Conclusions and Clinical Relevance—By use of a 2.7-mm rigid endoscope, liver biopsy procedures can be performed safely, swiftly, and easily in green iguanas. Biopsy specimens obtained by this technique are suitable for histologic examination. For evaluation of the liver and biopsy specimen collection in lizards, endoscopy is recommended. (J Am Vet Med Assoc 2007;230:1849–1853)

specimens.4,5 Compared with wedge tissue samples col-lected surgically, large-gauge–needle biopsy specimens yielded a diagnosis in only 48% of cases, and findings from examination of the latter should be interpreted with caution.6 Although the diagnostic value of surgical biopsy procedures is greater than that of needle biopsy and fine-needle aspiration techniques, the invasiveness of those procedures is also greater; however, that disad-vantage has been largely overcome since the develop-ment of minimally invasive endoscopic techniques.3,7,8 Retrospective studies9,10 in humans have revealed com-parable diagnosis rates for laparoscopic and laparoto-my-associated liver biopsy procedures, but application of endoscopy has the advantage of decreased duration of hospitalization.

In reptile medicine, the diagnostic approach to liver disease is similar to that of domesticated animals, and typically, results of an examination of liver tissue samples are required for a definitive diagnosis.11 Large-gauge–needle biopsy is seldom performed in small rep-tiles because of the dangers of iatrogenic trauma, and ultrasound-guided fine-needle aspiration frequently yields specimens from which a diagnosis cannot be made because of the difficulties of microscopic inter-pretation without tissue architecture. In snakes that weigh > 1 kg (2.2 lb), percutaneous ultrasound-guided needle biopsy has provided diagnostic samples; how-ever, 2 to 4 attempts were required to obtain liver tis-sue, and penetration of the gastrointestinal tract was a

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1850 Scientific Reports: Original Study JAVMA, Vol 230, No. 12, June 15, 2007

complication.12 The failure of the procedure was associ-ated with movement of the snake, and anesthesia was therefore deemed essential. Endoscopic liver biopsy has been advocated for a variety of reptile species, even in animals that weigh ≤ 100 g (0.22 lb).13-16 Anesthesia is required, but to our knowledge, no complications have been reported with use of appropriate anesthetic tech-niques. The purpose of the study reported here was to establish a safe and effective endoscopic technique for collection of liver biopsy specimens from lizards by use of a 2.7-mm rigid endoscope system that is commonly available in zoologic veterinary practice.

Materials and Methods

Animals—The study protocol was approved by the University of Georgia’s Institutional Animal Care and Use Committee (IACUC No. A2003-10074-m2). Eleven healthy subadult male green iguanas (Iguana iguana) were maintained in conditions approved by the Association for Assessment and Accreditation of Laboratory Animal Care. The iguanas were housed individually and maintained in a room with an ambient temperature of 23.8ºC (75ºF) at night and 27.2ºC (81ºF) during the day. A mercury-halide incandescent lamp suspended above each enclosure pro-vided a daytime basking area (35ºC [95ºF]) and broad-spectrum lighting. The iguanas were exposed to cycles of 12 hours of light followed by 12 hours of dark. The diet consisted of commercial iguana pelletsa soaked in water and supplements of several varieties of lettuce, col-lard greens, cabbage, and kale; water was available at all times. General environmental humidity was maintained at 80% through daily spraying. The iguanas were physi-cally examined on arrival and acclimatized to the research facilities for 7 days prior to the start of the study. Food was withheld from all 11 iguanas for 48 hours prior to anes-thesia, although access to water was maintained.

Anesthesia—Iguanas were accurately weighed and received butorphanol (1 mg/kg [0.45 mg/lb], IM) 20 minutes prior to induction of anesthesia with propofol (10 mg/kg [4.5 mg/lb], IV). Following intubation, anes-thesia was maintained by use of isoflurane in oxygen, ad-justed to the requirements of each iguana, and delivered via a pressure-cycle ventilator.b The risk of development of hypothermia was reduced by maintaining the anesthe-sia and surgery areas at 23.8ºC and placing the iguanas on recirculating warm water blankets. Anesthetic depth was monitored by evaluating reflexes, heart rate, end-tidal CO2 concentration, peripheral pulse, and oxygen saturation (as measured by pulse oximetry).

Surgery—Iguanas were positioned in left lateral re-cumbency (dorsum facing the surgeon) on a level surgery table (Figure 1). Following aseptic preparation of the right flank, a 3-mm vertical skin incision was made in the center of the paralumbar region. Then, with the surgeon pinch-ing the skin and underlying external oblique muscula-ture, a 4.8-mm (14.5-F) operating sheath with obturatorc was inserted through the skin incision, directed craniad through the external abdominal oblique musculature, and placed into the coelomic cavity. The obturator was removed and replaced with a 30o telescoped that was con-nected to an endoscopic video systeme (including camera,

monitor, xenon light source, and CO2 insufflator); CO2 insufflation flow and pressures were set to 0.5 L/min and 3 to 5 mm Hg, respectively. Once the coelom was inflated, the liver was identified and the entire right liver lobe and gallbladder were examined. By use of 1.7-mm (5-F) bi-opsy forcepsf inserted down the instrument channel of the operating sheath, 3 liver biopsy specimens were collected from the caudal edge of the right liver lobe. Tissue samples were transferred from the forceps to a histology cassette by use of a sterile cotton-tipped applicator moistened with sterile saline (0.9% NaCl) solution and then immediately placed in neutral-buffered 10% formalin. Following the manual expression of coelomic CO2, the telescope and sheath were removed and the skin incision was closed by use of 3-0 polydioxanone suture in a horizontal mattress pattern. Certain intervals were recorded to the nearest sec-ond as follows: time from initial skin incision to insuffla-tion and first clear observation of the liver, time from first clear observation of the liver to completion of the exami-nation of the right lobe and gallbladder, time from start to completion of collection of all 3 liver biopsy specimens, and time from start of CO2 expression from the coelom to completion of skin incision closure. Duration of anes-thesia was defined as the time from propofol injection to return to spontaneous respiration following cessation of isoflurane administration and was measured to the nearest minute. In addition, any complications associated with the anesthesia or surgical procedures were recorded. Af-ter recovery from anesthesia, the iguanas were returned to their enclosures. General behavior and food intake were monitored for 10 days.

Necropsy and tissue sample processing and ex-amination—Ten days following surgery, all iguanas were weighed and then euthanatized via IV administra-tion of pentobarbital sodium. Each iguana underwent a full necropsy examination. The liver was evaluated for any evidence of trauma or disease; liver tissue samples (along with any other tissues of abnormal appearance) were collected for histologic examination. Biopsy speci-mens and tissue samples obtained during necropsy were processed routinely, embedded in paraffin, sectioned at 5 µm, stained with H&E, and examined microscopi-cally. For each biopsy specimen, the degree of crush ar-tifact (ie, damage resulting in an inability to recognize

Figure 1—Illustration of an endoscopic liver biopsy procedure in a green iguana (Prepared by Kip Carter; printed with permission from Educational Resources, College of Veterinary Medicine, University of Georgia, Athens, Ga).

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cell types or evaluate the hepatic parenchyma) in each section was graded as follows: minimal, ≤ 10% affected; mild, 11% to 20% affected; moderate, 21% to 50% af-fected; and severe, ≥ 51% affected. The number of por-tal triads included in each section was also counted.

Results

Before surgery, the mean ± SD iguana body weight was 441 ± 42 g (0.97 ± 0.092 lb). Complete endoscopic examination of the right liver lobe and gallbladder and liver biopsy procedures were com-pleted successfully and without complication in all iguanas (Figure 2). The mean time from initial skin incision to insufflation and first clear observation of the liver was 95 ± 31 seconds. Time from first clear observation of the liver to completion of the exami-nation of the right lobe and gallbladder was 44 ± 9 seconds. Time from start to completion of collection of all 3 liver biopsy specimens was 209 ± 57 seconds, and time from start of CO2 expression from the coe-lom to completion of skin incision closure was 57 ± 17 seconds. Duration of anesthesia and surgery was 24 ± 7 minutes and 6.8 ± 1.0 minutes, respectively. Recovery from anesthesia was complete and unevent-ful in all iguanas. All iguanas returned to apparently normal behavior and feeding patterns by the day following surgery; behavior and feeding remained unchanged until the time of euthanasia 10 days af-ter surgery. At that time, the mean body weight was 449 ± 54 g (0.988 ± 0.119 lb).

At necropsy, there was no clinically important evidence of trauma or disease associated with the skin or muscle entry sites, liver, or any other viscer-al structures in any iguana. In 4 iguanas, 1 or more

small (1- to 2-mm-long) fibrin tags were detected at the endoscopic biopsy sites. In the remaining 7 igua-nas, there was no gross evidence of the endoscopic biopsy sites.

Three liver biopsy specimens from each iguana were examined histologically (33 evaluations overall). Crush artifact did not affect > 50% of any tissue sec-tion. Among the 33 biopsy specimens, sections of 8 (24.2%) had minimal crush artifact (Figure 3), sec-tions of 11 (33.3%) had mild crush artifact, and sec-tions of 14 (42.4%) had moderate crush artifact. In each instance, the crush artifact was confined to the periphery of the section and there was a central area of intact and undamaged parenchyma that could be eval-uated histologically. The mean number of portal tri-ads per section was 1.96. Triads were more frequently observed in sections with minimal crush artifact (2.9 triads/section) and mild crush artifact (1.9 triads/sec-tion) than in sections with moderate crush artifact (1.5 triads/section). No triads were apparent in 3 sec-tions with moderate crush artifact; triads may have been present but unrecognizable in these sections.

Figure 2—Representative views obtained during an endoscopic liver biopsy procedure in a green iguana illustrating the ventrolateral as-pect of the right liver lobe (A), gallbladder and caudal edge of the right liver lobe (B), the caudal edge of the right liver lobe during biop-sy specimen collection by use of 1.7-mm biopsy forceps (C), and the liver after completion of the biopsy procedure (D). For orientation, dorsal and ventral aspects of views A and B have been identified (d and v, respectively).

Figure 4—Photomicrograph of a section of a liver biopsy speci-men obtained endoscopically from an iguana. Mild hyperplasia of the biliary ductules is apparent. H&E stain; bar = 50 μm.

Figure 3—Photomicrograph of a section of a liver biopsy speci-men that was obtained endoscopically from an iguana. The speci-men has minimal crush artifact, which is confined to the periph-ery of the section. H&E stain; bar = 200 μm.

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Mild hyperplasia of the biliary ductules was detected in 1 biopsy specimen obtained from 1 iguana (Figure 4), and this finding was confirmed via histologic ex-amination of the liver tissue specimen obtained at nec-ropsy. Histologic abnormalities were not detected in any other biopsy specimen sections or sections of liver tissue collected at necropsy.

Discussion

The minimally invasive liver biopsy proce-dure described in this report was based on ac-cepted endoscopic methods for reptiles, and for iguanas in particular.13,14,16 Endoscopic liver bi-opsy was safe, simple to perform with appropri-ate equipment, and yielded tissue samples suitable for histologic interpretation in the present study. Coelioscopic examination (performed from the right side) provided excellent views not only of the right liver lobe and gallbladder, but also of portions of the gastrointestinal, urogenital, and cardiorespiratory systems. However, if the liver was the sole organ of interest in a particular iguana, a ventral approach (performed with care to avoid the midline abdominal vein) would permit evaluation of both the left and right liver lobes.

In small reptiles, ultrasound-guided fine-needle aspiration of the liver is possible but seldom recom-mended because practitioners are typically less familiar with their anatomic features; thus, the risk of iatrogenic damage resulting from the procedure is increased. In addition, cytologic interpretation of aspirates from rep-tiles is often difficult; therefore, there is a preference for histologic samples that preserve tissue architecture. En-doscopy and needle biopsy both require that the patient is anesthetized and yield biopsy specimens of compa-rable histologic quality; however, endoscopy may be preferable because direct observation of the organ of interest during sample collection reduces the risk of iatrogenic trauma.12,13,16

The size of the biopsy specimen is dictated by the size of the endoscopic forceps used. The expected tissue volumes from 1-mm (3-F), 1.7-mm (5-F), 3-mm (9-F), and 5-mm (15-F) biopsy forceps would be 0.5, 2.4, 14.1, and 65.4 mm3, respectively. Whereas the biopsy specimens collected from iguanas in the present study were small (2.4-mm3 volume), they were considered appropriate for small-sized iguanas (mean weight, 441 g) and did yield tissue that was suitable for histologic examination and histopatho-logic interpretation. In larger reptiles, the use of 3- or 5-mm forceps may be preferable, and a 1-mm in-strument may be better suited for use in reptiles that weigh < 100 g.

Although retrospective human studies9,10,17 have revealed that evaluation of liver tissue collected dur-ing endoscopy is associated with a diagnosis rate for liver disease of nearly 98%, such data are current-ly lacking for reptiles. In the present study, biliary hyperplasia was correctly diagnosed in 1 iguana via examination of biopsy specimens and confirmed via examination of tissue specimens obtain during nec-ropsy. However, with regard to liver disease among iguanas or other small reptiles, further research in-

volving comparisons between biopsy specimens ob-tained endoscopically and tissue samples obtained during surgery or necropsy is needed before the di-agnostic capability of coelioscopic techniques can be definitively determined. Our clinical experience with a wide variety of reptile species has indicated that en-doscopic collection of visceral biopsy specimens can be of considerable benefit; in the face of equivocal clinico-pathologic results, examination of those tissue samples often enables a diagnosis to be made.18-20 Overall, it appears that endoscopic liver biopsy in iguanid lizards can be recommended for the collection of tissue sam-ples that are suitable for histologic interpretation.

a. Adult iguana diet, Ziegler Bros Inc, Gardners, Pa.b. VT-5000, small animal ventilator, BAS Vetronics, Bioanalytical

Systems Inc, West Lafayette, Ind.c. 67065C, operating sheath for 64018BSA telescope, 14.5-F outer di-

ameter, Karl Storz Veterinary Endoscopy America Inc, Goleta, Calif.d. 64018BSA, autoclavable Hopkins rigid telescope, 2.7 mm X

18 cm working length, 30o, Karl Storz Veterinary Endoscopy America Inc, Goleta, Calif.

e. 69235106, veterinary video camera II, 9219-B Sony medical grade monitor, 201320-20 xenon light source, 26012C CO2 insufflator, Karl Storz Veterinary Endoscopy America Inc, Goleta, Calif.

f. 67161Z, flexible biopsy forceps, 5-F X 34-cm, Karl Storz Veteri-nary Endoscopy America Inc, Goleta, Calif.

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view of the quality of liver biopsy specimens. Am J Clin Pathol 2006;125:710–721.

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3. Richter KP. Laparoscopy in dogs and cats. Vet Clin North Am Small Anim Pract 2001;31:707–727.

4. Roth L. Comparison of liver cytology and biopsy diagnoses in dogs and cats: 56 cases. Vet Clin Pathol 2001;30:35–38.

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6. Cole TL, Center SA, Flood SN, et al. Diagnostic comparison of needle and wedge biopsy specimens of the liver in dogs and cats. J Am Vet Med Assoc 2002;220:1483–1490.

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A review of reptile diagnostic coelioscopy. J Herpetol Med Surg 2005;15:16–31.

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18. Hernandez-Divers SJ, Stahl S, Stedman NL, et al. Renal evalua-tion in the green iguana (Iguana iguana): assessment of plasma

biochemistry, glomerular filtration rate, and endoscopic biopsy. J Zoo Wildl Med 2005;36:155–168.

19. Hernandez-Divers SJ. Endoscopic renal evaluation and biopsy in chelonia. Vet Rec 2004;154:73–80.

20. Hernandez-Divers SJ, Shearer D. Pulmonary mycobacteriosis caused by Mycobacterium haemophilum and M marinum in a royal python. J Am Vet Med Assoc 2002;220:1661–1663.

EXOTIC ANIMAL ENDOSCOPY

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