Studies of the microcirculation using transparent tissue observation chambers inserted in the hamster cheek pouch

Transcription

1 J. Anat,. (1966), 100, 4, pp With 10 figures Printed in Great Britain Studies of the microcirculation using transparent tissue observation chambers inserted in the hamster cheek pouch By I. A. SEWELL* Westminster Medical School, London, S.W. 1 The ideal preparation for the study of the microcirculation must possess anatomical and physiological normality, at least during the initial stages of observations. It should be thin enough to enable the use of high powers of magnification where transmitted or reflected light is required. Repeated observations must be possible, preferably without anaesthesia. In some instances, direct accessibility to the preparation for biopsy may also be required. Whatever the method, minimal surgical interference is desirable to ensure preservation of the environment. None of the methods currently used satisfy all these criteria and the choice of the method will be determined by the most critical factor in the problem under investigation. The limitations of the chosen technique will have to be accepted, unless another method is used concurrently to overcome certain disadvantages peculiar to the first-chosen procedure. Thus, a thin preparation with relative permanence, and which can be easily handled for microscopic observation, is obtained by insertion of transparent tissue observation chambers into rabbits' ears (Sandison, 1924, 1928; Clark Kirby- Smith, Rex & Williams, 1930). But, the connective tissue which forms in the chamber is the result of a wound healing process and the vessels formed cannot be regarded as normal. Eversion of the cheek pouch of the hamster provides a readily accessible preparation which is normal in structure and function at the outset (Fulton, Jackson & Lutz, 1947; Lutz & Fulton, 1954; Fulton & Lutz, 1957), but it is rather too thick for higher powers of magnification. This can be overcome to some extent by dividing the outer layer of the exteriorised pouch at its summit and stripping off some of the fibrous tissue between the everted layers, but such a method reduces the procedure to a single acute experiment. This limitation also applies to exposing mesenteries for microcirculatory studies (Zweifach, 1948, 1954). Recently, Sanders (1963), Sanders & Shubik (1964), and Goodall, Sanders & Shubik (1965) have devised a method whereby the advantages of the hamster cheek pouch preparation can be combined with some of those of the transparent tissue observation chambers inserted into rabbits and mice (Algire, 1943). These workers inserted a specially designed transparent tissue observation chamber into the cheek pouch of a golden hamster so that the epithelial lining and overlying fibrovascular layer were included in the chamber cavity. Basically, this method provides a useful compromise of ideals. At the outset, there is anatomical and physiological normality. The chamber provides a relatively permanent preparation which allows for immediate and repeated observation. Unfortunately, these methods have not completely solved the problem of tissue thickness, and direct access to the tissue within the chamber is difficult. * Present address: Glasgow Royal Infirmary.

2 840 I. A. SEWELL This paper describes further development in design of transparent tissue observation chambers for insertion into the cheek pouch of a golden hamster. The basic design is essentially a modification of the chamber described by Goodall, et al. (1965) and has been used to facilitate the study of microcirculatory responses to materials used in clinical surgery (Sewell, 1966) to observe the effects of pharmacological substances on graft reactions, and the survival of implanted tissues. The techniques for insertion of the chambers described by Sanders and his colleagues have been modified and some improvement has been achieved in methods of transilluminating the tissues under observation for both microscopy and photomicrography. Dissected membrane Transparent observation window Monomer-polymer seal Chamber lid Base plate\ Base-plate pillar jaceiakw. A_. Plamysma muscle Lumnen Fibro-vascular.iyer If O>oC Fibro-areoar tissue Medial wall Fig. 1. Lining epitheliumn Transverse section through hamster cheek pouch to show anatomical relations of inserted transparent tissue observation chamber. The Westminster transparent tissue observation chambers The chambers so far developed are all smaller in dimensions and weight than those described by Sanders and his colleagues. The components common to all are a base plate, which lies within the cavity of the cheek pouch, a chamber lid containing an observation window, and a chamber lid cover which is fixed to the chamber lid to prevent the animal from damaging the observation window with its hind legs when not under observation. The lid is secured to the base plate by passing the bolts or pillars fixed to the latter through slits in the membrane, and then into holes in the chamber lid, securing with nuts or plastic gluing respectively. The lid is secured in the gap of the wound made to expose the cheek pouch in situ. The relations of the inserted and assembled chamber are illustrated in Figs. 1 and 2. A variety of plastic materials has been employed in the construction of the chambers. Of these, methacrylate and polystyrene have given the most satisfactory results. Both of these substances can be gamma-irradiated without serious effects on structure and stability, and this is the method used for sterilization prior to insertion.

3 Transparent chamber studies of the microcirculation 841 Westminster Mark I chamber This is the basic design upon which all other modifications are patterned. It is very simple and can be constructed in the laboratory workshop. It can be adapted to mass production methods using jigs or by injection-moulding techniques. The working drawings are shown in Fig. 3. Base Plate. This is a plastic disc of 1 mm thickness. The sharp edge of the surface which will lie in relation to the lumen of the pouch is cut and polished until it is smoothly convex to ensure a minimum of trauma to the surface of the medial wall of the cheek pouch. Two bolts of titanium are let into the base plate with their centres 4I'~~~~~~~~~~~ Transparen t-tissue observation chamber Ostium of cheek pouch [ Scapula Light train (transilluminator) Fig. 2. Position of inserted transparent tissue observation chamber, with light train (transilluminator) in position. lying on a circle of 21 mm diameter and their axes maintained exactly at a right angle to the plane of the base plate. Six holes of diameter of 2 mm are cut right through the plate with their centres lying on a circle of diameter 17 mm: they allow for the escape of fluid and desquamated epithelium from the surface of the membrane enclosed in the chamber. On the membrane surface of the chamber, two grooves measuring 6 mm in length, 1 mm wide and 05 mm in depth are cut with their long axes on a radius at 450 to the diameter of the plane which passes through the centres of the bolts. Chamber lid. This is a plastic ring 2 mm in thickness with an external diameter of 26 mm and an internal diameter of 18 mm. The outer margin is grooved to a depth of 1-5 mm to accommodate the edges of the incision in the skin when the chamber is finally assembled. Two holes are cut to allow the bolts of the base plate to pass through: these lie with their centres on a circle of diameter 21 mm. The observation window consists of a sheet of gauge 25 polyethylene terephyhalate (Melinex-I.C.I.).

4 842 I. A. SEWELL This is secured to the inner aspect of the chamber lid by applying a plastic adhesive while the window sheeting is held taught: the most satisfactory adhesive is Evostik S.H. 105 (Evode Ltd). Once the plastic adhesive is firm, the window sheeting covering the bolts is perforated and slots are also cut into it over the two drainage grooves: these slots are terminated 2 mm from the inner margin of the chamber External surface mm -' of chamber lid E-e21 I mm -t '.0 18 mm II Fig. 3. Irrigation i 17 mm--i groove _ Westminster Mark I transparent tissue observation chamber. Constructional details. _ lid ring. These drainage grooves allow for excess of fluid on the dissected surface of the membrane to drain away should pressure rise within the chamber cavity. Chamber lid cover. This consists of a very thin plastic disc which matches the outer margin of the chamber lid and has holes cut through it to accommodate the base plate bolts. It is conveniently made from 100 or 150 gauge polyethylene terephyhalate film. Bolts and nuts. As previously indicated, these are ideally of titanium, but stainless steel, brass and various alloys have been relatively successfully employed.

5 Transparent chamber studies of the microcirculation 843 Currently, size 8 B.A. threads are used for reasons of economy, but sizes 9-12 B.A. have been used at different times. The shaft length of the bolt must be at least 6 mm but should not be more than 9 mm. The securing nut which finally establishes the stability of the assembled chamber must be as thin as possible, but the gallows plate nut should be sufficiently broad to allow for easy manipulation (Fig. 3). Outer surface Line of section through chamber lid nut and bolt (B) Inner surface Line of section through (D) 7,injection tubing (C) Line of section through holes > Plastic spacers (E) for base-plate pillar (A) ((0 05 mm thick),' \\ ',Line of section through (C) drainage grooves (D) Section through lid on (Bm 2 line of base-plate pillars (A)8 -Ad Plastic spacer (E) ~~I-- 17 mm - \ Observation window 21 mm- Base-plate pillar 26mm Fig. 4. P C (B) M (D) >- Alloy bolt Drainage Siliconed flexible (B B.A.) groove nylon portex tubing (size 00) Westminster Mark II transparent tissue observation chamber. Constructional details. Westminster Mark II chamber This is a modification of the Mark I pattern, just described, to permit repeated injection of fluids into the chamber cavity and to accommodate varying sizes of implant. The working drawings for this pattern are shown in Fig. 4. The essential modifications are: Base plate. The bolts have been replaced by plastic pillars of diameter 2 mm, projecting 2-1 mm above the membrane surface of the plate. These plastic pillars have the advantage of remaining more securely and accurately in position during manipulation of the chamber at insertion, and during subsequent microscopic observations. But, final assemblage of the chamber requires them to be glued to the

6 844 I. A. SEWELL chamber lid, thus preventing salvage of the components when the experiment is terminated. Chamber lid. This has a reduced internal diameter of 17 mm. The bolt holes of the Mark I design have been widened to accommodate the plastic base plate pillars. Two lengths of siliconed nylon tubing have been let into the ring to provide for injection of fluids into the chamber cavity. Portex tubing has been found to give the most satisfactory results. In the drawing, size 00 is shown, but larger sizes may be used. To maintain the internal sterility of the chamber, the outlet of the tubing is stoppered as shown. Two metal bolts are also let into the chamber lid ring: these secure the chamber to the gallows plate during microscopic observation videe infra) and also the chamber lid cover in position when experiments are not in progress. Spacers. These are small rings of plastic, approximately 0 05 mm in thickness, with an external diameter of 3-5 mm. Their internal diameter is 2-1 mm so that they can be easily slipped over the base plate pillars to increase the capacity of the chamber cavity. They can be used in multiplicity, as well as singly. Chamber lid cover. This differs only from that described for the Mark I pattern in that holes for both chamber lid bolts and for the injection tubing are cut. Westminster Mark III chamber This is essentially similar to the Mark II pattern, but the bolts running through from base plate to lid for both security of the chamber and attachment to the gallows plate as described for the Mark I pattern have been used. Westminster Mark I V chamber This is basically the same as the Mark I pattern but its overall external diameter is 28 mm because the outer lip of the groove in the ring of the chamber lid overhangs the inner by 1 mm. The chamber lid is made of Alkon (an acetate co-polymer -I.C.I.) which is opaque enough for identification numbers to be written on to it in ink or pencil. All the components of this design can be cheaply and quickly turned out on a commercial basis. Preparation of the components of the transparent tissue observation chambers prior to insertion in the hamster On delivery the components have been found to be contaminated with dust and debris consequent on machining. Before the observation window is affixed, the chamber lid ring and the base plate are immersed in 10 % sodium hydroxide solution for 1 h and then transferred successively to three changes of ion-free water allowing 30 min for each change and ensuring that the final change remains at between ph 7 and 7*6 at the end of the immersion period. Clearance is then completed by drying in an oven at 37 0C for a further 30 min. No change in the metal bolts has been noticed following this procedure. The base plate bolts may be secured by applying a minute drop of plastic adhesive to the last thread and then screwing the bolt back into the base plate: Evostick SH 105 thinned down with acetone has been found suitable for the purpose. The observation window is now fixed to the chamber lid in the manner described in the account of the Westminster Mark I chamber videe supra). It must not be

7 Transparent chamber studies of the microcirculation 845 attempted prior to cleansing the parts with sodium hydroxide, otherwise clouding of the window may occur and the adhesive may be loosened. The components of the chamber are now checked for their integrity by mock assemblage. The parts are then packed separately in small polythene bags for sterilization by gamma-irradiation. This is the only reliable method: it does not produce physical or chemical changes in the components nor has any residual radioactivity been discovered. This procedure can be arranged through the United Kingdom Atomic Energy Authority Isotope Research Division at Wantage, or through Johnson's Ethical Plastics Ltd. at Slough: both organizations have undertaken to provide this as a free service for research workers. Selection, preparation and maintenance of the hamsters Golden hamsters of either sex, between 20 and 35 weeks of age, and weighing between 100 and 160 g were found most suitable. The presence of the inserted chamber did not materially interfere with their normal activities: it did not prevent gestation nor rearing of the young by a small group of female hamsters. Following operation, the hamsters were kept in separate plastic cages which were painted black, excluding light, to maintain an environment familiar to the animals. Separation was necessary because the presence of a chamber incited more fights between animals than usual. Unless nutrition was carefully controlled, it was found that insertion of a cheek pouch chamber produced weight loss until the chamber was extruded. This loss of weight could be reduced or delayed by supplementing the standard hamster dry feed pellets with an abundance of fresh green foods, fruit, fresh milk, and commercially prepared vitamin mixtures. The animals were isolated in the cages intended for the post-operative period on the day before operation. All the bedding was removed and a wire mesh was put down on the floor with sufficient clearance to allow the faeces to drop well out of reach of the animals. Hamsters are sometimes coprophagists, and faecal contamination of the cheek pouch will produce early necrosis of the membrane enclosed in the chamber. The animal was starved of solid food for at least 6 h prior to operation but water was allowed ad lib. Technique for insertion of the hamster transparent tissue observation chamber Anaesthesia. Both sodium pentobarbitone and urethane can be used as anaesthetics, but for most of the procedures on which this paper was based. the former was used. Orland & Orland (1946) give a full account of pentobarbitone anaesthesia in Syrian hamsters. The most satisfactory results were obtained with 2 % solutions of freshly prepared sodium pentobarbitone made up from the powder supplied for clinical use. 0*6 ml per 100 g body weight was given intraperitoneally prior to shaving the skin and preparing the cheek pouch. A further 0-2 ml was given irrespective of body weight immediately prior to actual operative procedures. Preparation of the operative field. An area from the base of the pinna extending medially to the mid-line and then caudally to the level of the inferior angle of the scapula and laterally to the fleshy root of the fore limb was shaved so that all the hair over half the neck and posterior wall of the thorax was removed (Fig. 2.).

8 \ I L _ X * ~~~~~~~~~~~~~~Elevation and 846 I. A. SEWELL Electric clippers with fine teeth were used, followed by close shaving with an open razor. All the loose hair was removed by swabbing with a detergent type of antiseptic such as domiphen and chlorhexidine in 80 % alcohol. When the area had dried, it was painted with tinct. benzoin co. (meth) to seal off bacteria laden crevices in the skin and to protect the wound edge postoperatively. Preparation of the cheek pouch. Under normal conditions, the cheek pouch contains food particles and fragments of the bedding straw. These must be removed, otherwise they will obscure part of the field during microscopy. The cheek pouch 22 cm 11 cm -i 9 mm tjjupper 2 aspect Position of base plate internal details \_, Lower aspect Fig. 5. Illuminating base-plate holder. Constructional details. A, Cystoscope lamp (GU Autoface/14 Charriere); B, platform; C, tongue (base plate grip); D, tongue pin; E, tongue pin fixing screw; F, tongue pin spring; G, shaft; H, channel for lamp leads. was gently everted by introducing a small dry cotton-wool swab on an orange stick through the mouth and into the ostium of the cheek pouch so that the epithelial surface adhered to the dry fibres. The cheek pouch following eversion was then spread out and thoroughly cleansed of all adherent particles using cottonwool swabs on orange sticks soaked in a weak aqueous solution of an antiseptic detergent. It was found that a warm 5 % aqueous solution of domiphen and chlorhexidine gave satisfactory results. Gentleness was found to be essential, otherwise damage to the epithelial surface caused injury, thrombosis and rupture of small blood vessels and also promoted epithelial desquamation in the postoperative period sufficient to obscure the microscopic field. Complete removal of debris from the lining of the cheek pouch was checked with a hand lens before the pouch was reposited. Insertion of the base plate. The previously gamma-irradiated base plate was removed from its polythene envelope by sterile non-toothed forceps and placed in a special base-plate holder illustrated in Fig. 5. The base plate rested on a platform or shelf which was illuminated by a small lamp (cystoscope bulb) lying immediately below the platform. The platform was attached to a shaft which carried a spring-

9 l _ow T 1 ~3 *5cm Transparent chamber studies of the microcirculation 847 loaded tongue or grip for holding the base plate firm during the operation for insertion of the chamber. A further development of the base-plate holder is in progress in which the platform is made of plastic that can be totally illuminated on the coldlight principle. The loaded base-plate holder was introduced into the cheek pouch in the following manner. The skin of the mouth was gently stretched by grasping with two pairs of guarded forceps until the ostium of the cheek pouch itself was clearly visible. With the utmost care the base-plate holder was then passed through the ostium until the 10 cm -- tt,- ~8-5cm * 21 b' smmi I Superstage ~~~17~ m I Gallows plate Vernier scale '0 ) 3 cm of microscope 1'_ mechanical stage Fixing screw4 Transparent tissue - observation chamber Flanged side of gallows plate Gallows-plate 7 pilars pi ~Pi-ism of 4 cm Superstage runner light train Cheek in mechanical pouch L -h stage groove 6 m Mechanical stage of microscope Superstage Fig. 6. Superstage and gallows plate. Constructional details (see also Figs. 7 and 9). base plate was judged to have reached the most caudal position possible without excessive stretching of the pouch. Failure to exercise due care could have ruptured the muscular sphincter guarding the ostium, resulting in subsequent discomfort to the animal due to prolapse of the chamber. Failure to advance the base plate sufficiently resulted in final emplacement too far cranially so that the animal found difficulty in feeding. This forward position also results in inclusion of that part of the membrane which is too thickly overlain with platysma muscle (Priddy & Brodie, 1948). These fibres pass very close to the fibrovascular layer and thus prevent a complete exposure of this part of the membrane. The summit of the pouch is also its more vascular part. m

10 A. SEWELL When in position in the cheek pouch, the base-plate holder was rotated until the base plate was judged to lie in an horizontal position. The pillars or bolts of the plate were adjusted to lie on an axis passing through the base of the pinna and the root of the fore limb. This is important where the gallows plate videe infra) has but one set of bolt holes drilled in it, but in the experiments described a plate with a multiplicity of holes was used (Fig. 6) and allowed for considerable latitude in fixation. Once correctly positioned, the shaft of the base-plate holder was firmly held in a bench clamp and the height adjusted so that the holder was well clear of the skull and thorax of the animal but did not lift the head completely off the working surface of the operating bench. Operative technique for insertion of the transparent chamber. (A film of this procedure (Sanders & Sewell, 1965) is available and can be borrowed from Dr A. G. Sanders, Sir William Dunn School of Pathology, University of Oxford.) All operative procedures were carried out using accepted clinical operative aseptic techniques. Caps, masks, protective aprons and sterile gloves were worn. The shaved surgical field was painted again with tinct. benzoin co. (meth.). The area of the incision was covered with a plastic adhesive drape (Band-Aid Sterile Drape- Johnson and Johnson Ltd.) and the operative field protected by draping with autoclaved paper towelling. The skin incision was made in the line of the base-plate bolts or pillars, being usually 2-5 cm in length. The skin and subcutaneous tissue over the inserted base plate were separated from the tissues covering the lateral wall of the cheek pouch by lifting with Graeffe's iris forceps and then undercutting by alternate blunt and fine dissection with straight, flat, blunt-nosed ophthalmic scissors until the margin of the base plate was reached. Care was taken not to cut across the fibres of the platysma muscle: these fibres were split to ensure their function postoperatively in retaining the chamber in position. With care, the skin and split platysma muscle were retracted over the margin of the base plate, the plastic adhesive skin drape protecting the deeper tissues from contamination with resident skin bacteria. Any remaining adherent platysma muscle fibres and the bulk of the fibro-areolar tissue were gently dissected free of the exposed membrane using the iris forceps and the ophthalmic scissors. There remained a fine layer of closely adherent fibroareolar tissue and some platysma muscle fibres in close association with the fibrovascular layer containing the blood vessels to be studied. This was cleared by picking up the fibres with Barraquer's corneal forceps and cutting them with Castroveijo's or de Wecker's iridectomy scissors. Great patience, even with considerable practice and skill, was required to clear the preparation sufficiently well enough to be of any value. In cases of doubt concerning sufficient clearance, the preparation was studied with a dissecting lens or binocular dissecting microscope, and dissection was continued under these conditions until no air bubbles or loose strands of tissue could be identified. The illuminated base-plate holder considerably facilitated this part of the procedure. Throughout the procedure, the preparation was kept moist with isotonic buffered solutions. In practice the best results were obtained with TC 199 (Morgan, Morton & Parker, 1950), but Hank's & Wallace (1949) balanced salt solution or Tyrode's (1910) solution were also found suitable. Two holes were then cut in the dissected membrane immediately over the base

11 Transparent chamber studies of the microcirculation 849 plate pillars or bolts, using the iridectomy scissors. Care was taken that the holes would just allow the passage of the pillars or bolts, and they were so positioned that the membrane was evenly spread in all directions without stretching. At this stage, all air bubbles between the membrane and the plate were gently pressed out. Where the purpose of the experiment was to study grafts or other implants, these were implanted at this stage. The chamber was then closed by fitting the chamber lid over the bolts or pillars. Using sterile forceps, the lid was carefully withdrawn from its gamma-irradiated polythene envelope and then positioned over the bolts or pillars whilst the membrane was gently irrigated and any air bubbles removed. If spacers were required (as with thick grafts) these were positioned over the pillars or bolts immediately prior to closure of the chamber. When in position, the lid was secured to the base plate by threading on the securing nuts but taking care not to tighten them so that vascular occlusion in the membrane blood vessels occurred. In chamber patterns where plastic pillars were substituted for base-plate bolts, security was obtained by either heating the plastic with a hot needle point to form a rivet, or by using a plastic glue. The wound edges were gently positioned around the outer circumference of the lid by using fine toothed tissue-holding foreceps. Care was taken to exclude any deep tissue being imprisoned between the edge of the wound and the groove in the outer circumference of the chamber lid. In most instances, correct assessment of the length of the incision ensured that the wound edges fitted closely to the chamber lid: the wound edges were sealed to the chamber lid with sterile methyl-2-cyanoacrylate (Eastman-Kodak Monomer 910-Ethicon Ltd). Where the skin edges lay loosely around the chamber lid, they were apposed with one, or at the most two, interrupted vertical mattress sutures using atraumatic nylon. The plastic adhesive drape was then trimmed to leave a margin of cm to act as an immediate postoperative protection to the wound. The grip or tongue of the base-plate holder was released and the holder gently withdrawn from the cheek pouch. Where anaesthesia was still adequate, microscopic observation was made immediately. Goodall, et al. (1965) advise against immediate postoperative microscopy but no untoward effects were noted in the presently reported experiments. Technique of microscopic observation Where microscopy did not immediately follow the insertion of a chamber, the hamster was anaesthetized as previously described. If a chamber lid cover had not been used, it was necessary to clear the observation window of debris. The pouch bearing the chamber was cleaned out very gently with swabs on orange sticks soaked in mild warm antiseptic detergent (domiphen and chlorhexidine), care being taken to avoid traction on the membrane around the margins of the base plate. The centre of the base-plate required particular attention since it lay directly in the path of the light train. A flat director made of perspex was introduced into the cavity of the cheek pouch to provide a smooth passage for the prism-carrying sleeve of the transilluminator videe infra) into the lumen of the cheek pouch. The animal was laid, with the chamber-bearing side uppermost, on a specially

12 A. SEWELL constructed superstage carrying a gallows plate (Fig 6, 7). The chamber lid was aligned so that the holes in the gallows plate threaded for the bolts were directly over these and could then be secured by applying the gallows-plate nuts. The superstage was then positioned on to the mechanical stage of the microscope by sliding the runner on its undersurface into the groove which normally carried the slide fixers. The superstage was finally secured by one or two turns of the fixing screw. Gallows plate X S k y, ~~~~~~~~Bolt holes Gallows-plate nuts Observation window nsmargin of transparent tissue observation chamber S u pe r-stage Margin of chamber lid well Fig. 7. Hamster in position on superstage with gallows plate nuts applied to chamber bolts. Transillumination of the preparation cannot be so easily achieved as with the rabbit ear chamber since the substage apparatus of the microscope is put out of action by the presence of the superstage and the body of the animal. Hence, the light train must be carried in through the mouth and bent through an angle of 900 at the inner surface of the chamber. A simple light train can be established by passing a beam of light down a square-sectioned perspex rod with the end in the cheek pouch mitred at 45 0 to act as a prism. The emergent light train cannot be focused and therefore, critical illumination is not possible. However, such illumination is satisfactory for inspection at low powers of magnification and many of the early photomicrographs were made by this method. For more critical studies, the light must be focused on the membrane within the chamber, and for this purpose, the transilluminator illustrated in Fig. 8 was devised: it is fully discussed elsewhere (Sanders & Sewell). The objective of the microscope and the prismatic component of the transilluminator must lie in the same optical plane. This was achieved by rigid fixation of both microscope and the

13 Transparent chamber studies of the microcirculation 851 transilluminator on the same axis. The loaded superstage was positioned on the mechanical stage of the microscope and secured by tightening the fixing screw. The prismatic component of the transilluminator was gently advanced along the plastic director into the cheek pouch of the hamster until the light beam was approximately Quartz iodine Collimator 1t-is diaphragm.ri I-A. Ias nt roi) tfield co Heat filters A// Passage through mouth into ostium of cheek pouch Objective of observation Gallows plate iris diaphragm Condenser lultropak objective) Fig. 8. The transilluminator. Principles of construction and application. Leitz UO -Gallows-plate Plastic director Mechanical stage of microscope Fig. 9. The transilluminator in position in the lumen of the cheek pouch. Note the use of plastic director to facilitate entry. in the centre of the chamber (Fig. 9). Preliminary inspection was made with a 3 x objective to facilitate final adjustment of the position of the superstage and transilluminator. Succeedingly higher powers of objectives were then used, but for magnifications above 10 x, the Leitz UO (Ultropak) series with annular condensers removed were required to accommodate the well of the chamber. Oil and water immersion objectives have been satisfactorily employed. 54 Anat. 100

14 852 I. A. SEWELL Photomicrography Both monotone and colour photomicrographs have been made using the microscopic method just described. The best results were obtained using an automatic camera head whereby the speed of the shutter was automatically determined by the measurement of light intensity through a photo-electric cell and the shutter device working by remote control. Good results were also obtained by using an interchangeable microscope camera head with preliminary assessment of light intensity by a standard laboratory photo-electric cell and galvanometer monitor. A more detailed account of these procedures is given elsewhere (Sanders & Sewell, 1966). The monotone photomicrographs were produced from either SP3 (Ilford), Pan F (Ilford) or Plus X (Kodak). Colour photomicrographs were taken as transparencies, using Kodachrome II KRA or Ektachrome EBH (Kodak), the light intensity of the transilluminator being adequate enough without having to use flash illumination. RESULTS The designs of the chamber described and the techniques employed for insertion, for microscopy and photomicrography have been studied on 150 golden hamsters. There were only three deaths, two of which were due to using sodium pentobarbitone solutions which had been prepared for too long prior to administration. No deaths resulted directly from the operative procedures described. The animals were not unduly handicapped by the presence of the chamber. During the first few days following insertion, some of the animals attempted to remove the chambers by vigorous scratching with the hind limb. but no animal was successful in this. Until the incorporation of the chamber lid cover (see description of Mark I pattern of chamber), the observation window was frequently scratched by the hind limb of the animal. The cheek pouch into which the chamber was inserted was utilized after operation in the normal manner. Evidence for this was provided by the necessity to remove food and debris from it prior to further microscopic studies. Again, female hamsters who had borne litters were observed to regularly place their pups in the pouch when disturbed. Insertion of the chambers had well-marked effects on the body weights of the animals. On a standard commercially recommended diet (Oxoid S.G. 1) all the animals lost weight. Loss of the chamber, either by removal for histological studies or by extrusion in time, resulted in regain of weight to pre-experimental levels. The weight loss was diminished by modifying the diet with fresh greenstuffs and vitamin supplements. It is considered that the loss of weight was due to a combination of the effects of local infection and the influence of isolation of the animals on their normal feeding habits: investigation of these problems is in progress. Following insertion of the chamber, the enclosed membrane remained viable for a varying length of time. When there was no implanted substance, the average time was d, with several remaining viable and useful preparations for as long as d. After these times, necrotic changes were observed to extend inwards from the margin, most commonly from the cranial end. Several days elapsed before the chamber was finally extruded.

15 Transparent chamber studies of the microcirculation 853 Fig. 10 Photomicrographs of the dissected lining of the hamster cheek pouch enclosed in the transparent tissue observation chamber. (A) Freshly dissected membrane, x 30 (B) Freshly dissected membrane, x 220 (C) Membrane after 28 days, x 30 (D) Membrane with human bladder carcinoma heterograft after 6 days, x

16 I. A. SEWELL 854 By the methods described above, anatomical and physiological studies of the microcirculation in the hamster have been made. Studies have also been made of the microcirculatory changes which result from implantation of the materials used in clinical surgery (Sewell, 1966). Investigations are in progress on the microvascular responses to homografts and heterografts of both normal and neoplastic tissues, on the cellular exudates which occur in response to connective tissue disturbance and on certain aspects of the clotting mechanism. In Fig. 10, a series of photomicrographs are shown to illustrate the scope of the methods described. DISCUSSION Advantage was taken by Sanders (1963) of the more commendable features of the rabbit ear chamber when he designed a special transparent tissue observation chamber for insertion into the cheek pouch of the hamster. The pathological nature of the connective tissue formed in the rabbit ear was eliminated by substituting the dissected lining of the cheek pouch of the hamster in which the microvasculature was normal in both structure and function. Since there was no longer any necessity for delay until connective tissue had formed, the preparation was immediately available for microscopic study. Implantation could be performed at the time of insertion of the chamber, thus removing the relative disadvantage of re-opening the chamber cavity. Such a method increases the scope for the study of microvascular changes because the small size of the animal increases the number that can be retained in the animal house and reduces the latent period of usefulness of the preparation. Even with the most careful and skilled dissection, the thickness of the preparation is a limiting factor where very high powers of magnification are required. Too enthusiastic a dissection leads to damage to blood vessels in the preparation with troublesome haemorrhage, thrombosis and closure of fairly large areas of the capillary bed. Illumination of the preparation calls for specially developed apparatus such as the illuminated base plate holder, the superstage and gallows plate, and the transilluminator. Unlike the rabbit ear chamber method, the hamster must always be anaesthetized for repeated microscopy. Although it is a reasonably permanent preparation on which repeated observations can be made, its period of usefulness is comparatively short. Notwithstanding the various disadvantages, Sanders (1963), Sanders.& Shubik (1964), Goodall, et al. (1965) and Sewell (1966) have demonstrated the potentiality of the method for investigating a wide range of problems. Where such limitations as the relative thickness of the membrane and the short life of the preparation can be accepted, the method would appear to offer a useful means of studying microvascular responses. So far, access to the preparation other than for fluid-borne therapeutic agents, has not been required: it is however possible to modify the design of the chamber to achieve this. Construction of the chambers requires skill in the handling of plastic materials and this may present a financial problem where they have to be made commercially because such workshop production requires time-consuming skilled labour. This can be partially overcome by ordering large numbers of components whereby the setting up of jigs becomes feasible. However, the number of research workers in

17 Transparent chamber studies of the microcirculation 855 this field is small, particular interests in the various aspects of microcirculation vary, and to date, no standard pattern of chamber to suit all needs has been evolved. It is hoped that the basic essentials in the Mark I and Mark IV patterns may partially solve this problem because their basic design allows for a variety of modifications to be made once their components have been delivered to the laboratory: the cost of the components of the Mark IV pattern are low. The relatively early onset of infection and the extrusion of the chamber due to the ensuing necrosis presents a problem. Personal communication with other workers in this field reveals that this is a common complication. It can be delayed by the daily administration of antibiotics, but it would also appear that much can be done to prevent it by strict attention to surgical asepsis during the insertion of the chamber, not only in respect of the operation field itself but also by carrying out such procedures in a well-equipped animal operating room devoted entirely to the purpose. Daily attention to animal welfare is essential and measures should be taken to prevent faecal contamination of the wound: removal of all bedding from the cage and the provision of a wire grill to prevent coprophagism all assist in reducing infection from this source. SUMMARY Modifications in the designs of transparent tissue observation chambers described by Sanders (1963), Sanders & Shubik (1964) and Goodall et al. (1965) for the study of the microcirculation in the lining of the cheek pouch of the hamster have been made so that a series of such chambers has been developed (Westminster Series) to extend the scope of such investigations. A basic design (Westminster Mark I pattern) is described in detail, from which other types in the series have been developed. These include a chamber which permits direct and continuous application of therapeutic substances into the chamber cavity (Westminster Mark II pattern) during microscopy. These designs can also be modified for mass-production (Westminster Mark IV pattern), thus effecting a considerable reduction in cost. Techniques for preparation of the chambers prior to assemblage are described and the procedure for insertion into the cheek pouch of the hamster is outlined. The problems of microscopic observations are discussed together with the use of a special transilluminating device. One hundred and fifty hamsters have been subjected to the methods described. Three deaths have occurred as a result of the anaesthetic procedure but none have been due to the operative technique. The methods have been used to make anatomical and physiological observations for both research and teaching purposes, and have been employed to study microvascular reactions to the materials used in clinical surgical procedures, to homografts and heterografts of both normal and malignant tissues, and in studies of some aspects of the clotting mechanism. None of the animals has been incapacitated by the presence of the chamber, although weight loss until extrusion was a regular feature. Grateful thanks for advice and skilled technical assistance are due particularly to: Dr A. G. Sanders (Sir William Dunn School of Pathology, University of Oxford),

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