India Ink Injection Research Articles

The three-dimensional microstructure of intramural lymphatics in the canine large intestine

The three-dimensional microstructure of the intramural lymphatics of canine large intestine was clarified micrographically. After direct injection of India ink into the colon wall, we prepared 2 less than 3cm full-thickness transparent specimens of the opened colon wall and transparent sections. Lymph vessels were distinguished from blood vessels by intraarterial injection of cinnabar ink. A stereomicroscope was used for observation, and stereograms were reproduced. Lymph vessel networks were present in the shallow and deep layers of the lamina propria mucosae and the deep layer of the submucosa. The lymph vessel networks in the lamina propria mucosae were present immediately beneath the blood capillary networks. The thickest lymph vessels and the densest network appeared in the submucosa. From the submucosa, some of the lymph vessels existed immediately to merge into the collecting trunks and other lymph vessels which compose a minor pathway existed along lymph vessels in the muscular layer and finally merged into the collecting trunks in the subserosa.

Vascular anatomy of the pronator quadratus muscle-bone flap: A justification for its use with a distally based blood supply

The vascular anatomy of the pronator quadratus muscle and distal radius was studied in 24 cadaveric forearms by dissection under magnification and india ink and latex injection studies. The anterior interosseous artery divides into a muscular branch and a dorsal branch 1 cm to 3.5 cm from the proximal margin of the pronator quadratus muscle, and on its deep surface. Injection studies showed a rich periosteal plexus contributed by the anterior interosseous artery. Retrograde injection through the dorsal branch also showed good perfusion of the distal radius that was confirmed microscopically on bone sections. A muscle-bone graft from the anteromedial cortex of distal radius with an intact anterior interosseous artery has mobility of less than 2 cm. After ligating and dividing the anterior interosseous artery, blood supply to the distal radius bone flap relies on a retrograde flow and the bone flap could then be mobilized distally up to 4 to 6 cm.

Deep partial skin thickness burns: A reproducible animal model to study burn wound healing

A reproducible deep partial skin thickness burn model using guinea-pigs to study the healing process of this injury is described. Round aluminium templates heated to 75°C and applied for 5 s to the moistened, clipped and depilated dorsal skin produced the desired depth of injury. This model is applicable for the study of the three main components of the burn wound healing process: epithelialization, contraction and scar formation. It is recommended that the India ink injection technique be used to confirm the depth of the burn wound.

Experimental lumbar spinal stenosis. Analysis of the cortical evoked potentials, microvasculature, and histopathology.

An animal model of lumbar spinal stenosis was developed in which the pathophysiology of this condition could be examined. Four experimental groups, each containing six dogs, were studied. One group had a laminectomy of the sixth and seventh lumbar vertebrae only; these animals served as controls. In the three other groups, a laminectomy was performed and the cauda equina was constricted by 25, 50, or 75 per cent to produce chronic compression. Cortical evoked potentials were recorded preoperatively, immediately after constriction, and at one, two, and three months postoperatively. Daily neurological examinations were carried out, and the neurological deficits were graded using the Tarlov system. After three months of constriction, the cauda equina of three dogs in each group was examined histologically, and the vascular circulation was examined by latex and India-ink injection with a modification of the Spalteholz technique. The animals in the control group showed no neurological abnormalities, no changes in cortical evoked potentials, normal microvascularity, and no histopathological changes in the nerve roots or the spinal cord. The dogs in which the cauda equina had been constricted 25 per cent had no neurological deficits, mild changes in cortical evoked potentials, slight histological changes, and venous congestion of the root and dorsal root ganglion of the seventh lumbar nerve. The dogs in which the cauda equina had been constricted 50 per cent had mild initial motor weakness, major changes in cortical evoked potentials, edema and loss of myelin in the root of the seventh lumbar nerve, and moderate or severe venous congestion of the root and dorsal root ganglion of the seventh lumbar nerve. The dogs in which the cauda equina had been constricted 75 per cent had significant weakness, paralysis of the tail, and urinary incontinence; two dogs recovered by the third month, but all had neurogenic claudication for three months. All six dogs had dramatic changes in cortical evoked potentials and had complete nerve-root atrophy at the level of the constriction. There was blockage of axoplasmic flow and wallerian degeneration of the motor nerve roots distal to the constriction and of the sensory roots proximal to the constriction, as well as degeneration of the posterior column. Severe arterial narrowing at the level of the constriction and venous congestion of the roots and dorsal root ganglia of the seventh lumbar and first sacral nerves were also present. Cortical evoked potentials revealed neurological abnormalities before the appearance of neurological signs and symptoms.(ABSTRACT TRUNCATED AT 400 WORDS)

An experimental study of healing of the partially severed flexor tendon in chickens

There is a lack of clinical and experimental studies of the treatment of incompletely transected tendons. The controversy concerning the source of flexor tendon nutrients is of important clinical concern in healing of the injured tendon; thus, the flexor tendon blood supply has cited as a reason for using specific tendon suture techniques, and as a rationale for preserving the superficialis tendon and its vincula during tendon repair surgery. Our knowledge of the normal physiology of digital flexor tendons and the mechanism of their healing process is deficient. The aim of this study was to investigate the relative importance of the synovial fluid and the blood supply respectively for the healing of partially severed flexor tendons. We observed the sequential histological and vascular changes which occur in healing of the partial lacerations in the dorsal and plantar aspects of the tendons. We observed the vascularities of the two partially severed tendon groups after injection of microfil and india ink through the femoral artery. In the healing process there was no sequential histological difference between the dorsal and the plantar severed tendons. The vascularity patterns of the healing tendons were significantly increased and the hypervascularity of dorsal severed tendons was greater than that of plantar severed tendons. Partially severed tendons were completely healed without surgical repair with dense collagen fibers without adhesion in most cases. We concluded from this study that the blood vessels appeared to play a significant role in the healing of the severed flexor tendons. An intact synovial environment did not seem to be required for healing of the severed tendon. It is not necessary to surgically repair the partially severed tendon for prevention of rupture and adhesion.

Regulation of air and blood flow through the booklungs of the desert scorpion, Paruroctonus mesaensis

Injections of dye, latex and India ink were used to reveal the path of hemolymph circulation through the scorpion booklungs. Fine, branched arteries carry blood directly to muscle and other organs. The blood returns through venous channels to the ventral mesosoma where it passes laterally through the booklungs and into the pneumocardial veins just beneath the pleural cuticle. Blood flows dorsally through these veins to the pericardial sinus and heart. The scorpion has four pairs of booklungs located in the anterior segments of the ventral mesosoma. Each booklung has a spiracle which opens into an atrium enclosed by cuticular membrane. Air passes from the atrium into the booklung lamellae. Agitation of the animal or application of CO 2 causes retraction of the anterior and posterior atrial membrane. This expands the atrial chamber and allows gas exchange in the booklung lamellae. The posterior atrial membrane has a specialized region which forms a springy valve. This normally closes the spiracle unless pulled open by contraction of the attached poststigmaticus muscle. The pectens and receptors within the atrium may mediate the responses to CO 2. Slender hypocardial ligaments containing muscle fibers extend from the heart (dorsal mesosoma) to the booklungs in the ventral mesosoma. Heart movements thus cause dorso-ventral movement of the booklungs. The significance of these movements is as yet unclear. They may increase ventilation, help force blood to the heart and/or agitate the blood and booklung lamellae and thereby aid gas exchange. Passage of blood through the booklungs is regulated by dorsal and ventral muscles attached to the atrium at the lateral edge of the booklung. Contraction of the ventral atrial muscle closes the excurrent channel for passage of blood from the booklung into the pneumocardial vein. Electrical stimulation of the segmentai nerves from the subesophageal and first three abdominal ganglia causes spiracle opening and contraction of muscles attached to the atrial membrane. A previous study showed that these same segmental nerves also modulate heart activity. They thus provide a major pathway for regulation of the respiratory and circulatory systems.

The vascularized saphenous nerve as cross-face nerve graft

The vascular anatomy of the saphenous nerve was studied by cadaver preparation, Indian ink and micropaque injection. A constant vascularized nerve graft with a good pedicle and suitable length for a cross-face anastomosis was found. The vascularized saphenous nerve was used for the first time as a cross-face graft in 8 patients with facial palsy. The Tinel-sign was followed-up and a biopsy of the nerve graft at the time of the muscle transplantation was carried out to compare the morphological results of the vascularized nerve graft with two free sural nerve grafts which were used in two patients at the same time. The functional results are demonstrated.

Effect of light on oxygen-induced retinopathy in the rat model

The purpose of this study was to establish whether exposure to intense lighting favors the development or aggravates experimental oxygen-induced retinopathy in the newborn rat. Five groups of Wistar rats were studied. The control group was maintained for the first 14 days of life under conditions of cyclical (12L:12D) lighting at 12 Lx in room air. Two other groups were subjected, for the same amount of time, to semi-darkness (2 Lx; 12L: 12D), one with room air and the other with supplemental 80% oxygen. The final two groups were exposed to the same room air and hyperoxic treatments under intense lighting conditions (600 Lx; 12L:12D). After the treatment period, four rats were randomly chosen from each group, sacrificed and their retinas examined under electron microscope. Marked structural changes were seen only in the photoreceptor outer segments of those rats exposed to intense light. In eighty-five of the remaining rats retinal vascular morphology was examined in retinal flat mounts after intracardiac injection of India ink. Retinopathy was observed in rats treated with hyperoxia but no significant differences could be attributed to the light conditions under which the retinopathic rats had been maintained. In the rest of the rats, axonal transport along the optical pathways was evaluated after intravitreal injection of (3H) taurine. In the two groups exposed to hyperoxia, axonal transport was altered, but less markedly in those exposed to intense lighting than in those exposed to semi-darkness. Intense illumination under conditions of normoxia favors axonal transport. Exposure to intense lighting does not seem to aggravate oxygen induced retinopathy in the rat though it does produce structural lesions of the photoreceptors.

Respiratory responses to occlusion or hypercapnic blood injection of the anterior inferior cerebellar artery in cats.

To examine whether the central chemoreceptors of respiration are located in the perfused area of the anterior inferior cerebellar artery (AICA), we occluded arteries or injected hypercapnic blood into arteries in the ventral surface of the medulla in anesthetized, paralyzed, and peripherally chemodenervated cats. Phrenic nerve activity, as an index of respiratory output, was augmented by an injection of hypercapnic blood into the vertebral artery. This vertebral-injection response decreased during bilateral occlusion of AICA. However, responses of phrenic nerve activity to the occlusion of AICA were complicated; activity increased in 19 cats, did not change in 10, and decreased in 9 during occlusion. In experiments with blood of various PCO2 levels being bilaterally injected into AICA, phrenic discharges increased with increases of PCO2. During the injection of constant PCO2 blood into AICA, phrenic response to alveolar PCO2 decreased by 80% compared with the original response. From these results, the blood flow and blood PCO2 level of AICA seemed to be related to the central chemosensitivity for respiration. To examine the perfused area, the ventral surface pH of the medulla was measured with a micro-combination pH electrode (2 mm diameter). During the injection into AICA, pH in the rostral medulla depended on the PCO2 of injected blood, and pH in other areas depended on the PCO2 of systemic blood. Also, histological study of India ink injection into AICA showed that ink-filled vessels were exclusively observed in the rostral medulla. Thus, we conclude that at least part of the central chemoreceptors of respiration are located in the perfused area of AICA, that is, in the rostral medulla.

Posterior half amputation of the chick wing bud: the response of the developing vasculature, and subsequent wound healing.

Experimental analyses examining pattern formation in the developing chick limb have concentrated on the skeleton, muscles and nerves, and have rarely considered blood vessels. To investigate the relationship between the vasculature and limb development, posterior amputations were performed on 3.5-4 day chick limb-buds. It has been shown that the removal of the posterior half alters the developmental fate of the anterior tissue: it becomes necrotic and fails to differentiate into the complement of skeletal parts predicted by fate maps. The possibility that this developmental failure results from interference with the future arterial supply was examined by Indian ink injection between 3-48 h after operation. Scanning electron microscopy (SEM) and resin histology were used to examine the wound repair at similar post-operative intervals. Results from the Indian ink injections showed that within 6 h of operation a collateral circulation was established by means of a branch from the truncated primary subclavian artery. The capillary density in the operated limbs appeared normal when compared to the contralateral limb. The results support the view that the poor developmental performance of the anterior half is due to removal of the zone of polarizing activity (ZPA) rather than to experimentally-induced alteration to the vascular supply. Histological and SEM examination of the wound healing process showed that epithelialization of the cut surface occurred within 24 h, and that the peridermal cells of the bilayered ectoderm appeared to initiate the regrowth. The wound site was not visible 48 h after operation, showing that wound healing at these developmental ages occurs quickly, with no scar tissue formation.(ABSTRACT TRUNCATED AT 250 WORDS)

Origins of the neurovascular bundle: interactions between developing nerves and blood vessels in embryonic chick skin.

Growth cones of nerves and endothelial cells of blood vessels are closely analogous in their migratory behavior, and they are both set a similar task during the early development of a limb. Both must invade the mesenchyme to form ramifying networks of large nerves and vessels. Both systems must densely pervade certain regions of the developing limb, such as muscle rudiments, and both form dense cutaneous plexuses at precisely the same depth beneath the epidermis. Moreover, adult tissues show many examples of neurovascular bundles in which nerves and blood vessels run closely parallel and branch in a correlated fashion, suggesting some interdependence during development. We have examined the interrelationship between developing nerves and blood vessels in chick wing skin because it allows a particularly convenient two-dimensional analysis of the two systems which can be revealed simultaneously in the same preparation by injection of Indian ink combined with silver-staining. We show that nerves do not use blood vessels as pathways along which to crawl, but that there are two other ways in which neurovascular associations arise: in some situations nerves and blood vessels follow the same route because they are responding independently to the same mesenchymal cues; and in some situations nerves induce blood vessels to remodel around them.

Neural crest ablation does not alter pulmonary vein development in the chick embryo.

Cranial neural crest, which extends from the mid-diencephalon to somite five, plays an integral role in development of pharyngeal arch derivatives and supplies mesenchyme to the aortic arch arteries. Neural crest cells in pharyngeal arches three, four, and six migrate to the heart and are involved in aorticopulmonary and conotruncal septation. Ablation of the "cardiac" neural crest cells in chick embryos results in a variety of outflow tract anomalies, including persistent truncus arteriosus. Although other studies have shown the importance of the neural crest in the development of the cardiac outflow tract, the role of neural crest in venous development has not been established. This investigation evaluates the effect of cardiac neural crest ablation on the morphological development of the pulmonary vein. The presence of the pulmonary vein was confirmed initially at early stage 15 using histological sections and computer reconstructions of serially sectioned, normal embryos. India ink injections demonstrated a complete, patent pulmonary circuit at stage 18. Cardiac neural crest was ablated at stages 8-10. Operated, sham-operated, and control embryos were sacrificed at incubation day 11, and acrylic plastic casts prepared of the intravascular compartment. In experimental embryos with persistent truncus arteriosus, there were no morphological differences in the pulmonary veins, compared with shams and controls. These data indicate that the lesions of the cardiac neural crest have little morphological impact on pulmonary vein development. It is concluded that alterations in the cardiac neural crest are not involved in venous anomalies such as cor triatriatum and total or partial anomalous pulmonary venous return.

Endoscopic clip-marking of lesions using the newly developed HX-3L clip.

Injection of India ink and clipping are relatively well-known methods for marking lesions of the gastrointestinal tract. The use of metal clips provides a radiologically recognizable mark, which is also palpable at surgery. In addition, clips can be extremely useful as landmarks in comparing endoscopic findings with the resected specimen. Previous reports of clip-marking have concentrated on the Olympus (Tokyo, Japan) HX-2L clip, but this clip has the disadvantages of having too wide a diameter to be used through most conventional endoscopes and also a relatively low clip-retention rate, as the depth of its bite is limited. Recently, Olympus has marketed a new clip (HX-3L) designed by the author for hemostasis in cases of gastrointestinal bleeding. This new clip is an improved version of the cassette-type J-clip that was previously reported by the author in this journal. The prime advantage of the HX-3L is that the endoscopist can use it via a panendoscope without any assistance. In addition, it has an excellent bite and a very high level of safety. In terms of function and application, it is completely different from the HX-2L. In addition, the tip (the portion that actually grasps tissue) of the HX-3L is longer and thinner than the HX-2L, thus permitting a firm grasp of the muscularis mucosae layer even when approaching from an upper oblique angle and this, in turn, improves the performance of the clip as a marker.(ABSTRACT TRUNCATED AT 250 WORDS)

The blood supply of the calcaneal tendon.

The microvascular anatomy of the calcaneal tendon was investigated in cadaver tendons by injection of barium sulphate and indian ink and a quantitative study of intratendinous blood supply was made, using a computer-assisted image analysis system. There was a reduction in both the number and the mean relative area of vessels in the mid-section of the tendon. This area of reduced vascularity may be of significance in the pathogenesis of rupture.

The anterior tibial vessels and their role in epiphyseal and diaphyseal transfer of the fibula: experimental study and clinical applications

The blood supply to the fibula from the anterior tibial and peroneal arteries was investigated in 66 fresh cadavar limbs by India ink injection and radiographic studies. These studies revealed a reliable blood supply to the proximal epiphysis and the proximal two-thirds of the diaphysis of the fibula from the anterior tibial artery. Although the peroneal artery provided the dominant supply to the shaft of the bone, its contribution to the head of the fibula was scanty. The cutaneous supply of each vessel was defined and correlated with the territories of the other vessels supplying the leg. The vascular anatomy of the leg was examined in 100 clinical angiograms, revealing 21 limbs with anomalies. This reinforces the case for preoperative angiogram studies where transfer of the fibula is contemplated. The technique of harvesting the proximal growth plate and the shaft of the fibula on the anterior tibial vessels alone is described and illustrated with two successful clinical cases, each followed for 4 years. The growth rate of the transferred epiphysis averaged 1 cm per year.

Direct, real time measurement of meniscal blood flow. An experimental investigation in sheep.

The functional microcirculatory system of the meniscus was investigated in nine sheep using laser Doppler flowmetry (LDF). The highest blood flows recorded were found at the periphery and at the anterior and posterior horns, and this finding was confirmed by india ink injections in five sheep and by meniscal autoradiographs following intraatrial injection of 20 million Cs46 microspheres in four sheep. LDF provides accurate and reproducible assessment of meniscal blood flow. If adequate probes can be developed, this method offers great promise in the clinical assessment of blood flow within the substance of meniscal tears, thus offering the surgeon initial information in the decision to repair or resect a meniscal tear.

Developmental studies on the rat vomeronasal organ: vascular pattern and neuroepithelial differentiation. I. Light microscopy

The origin and the developmental sequence of the rat vomeronasal organ and its vascular supply are followed by means of India ink injection in serial sections of celloidin-embedded embryos from the eleventh day of gestation up to birth. The anlage of the vomeronasal organ has been established by the twelfth day of gestation (E 12). It appears as a shallow longitudinal impression of the medial wall of the nasal pit. At day E 14, it separates from the epithelium of the primary nasal cavity, forming a tube. The lumen of the organ remains continuous with the nasal cavity frontally, but ends blindly at the edge of the primary palate dorsally. From day E 16 to E18 the lateral surface of the tubular vomeronasal organ invaginates toward the lumen forming a wide longitudinal furrow. The lumen is bordered by the developing neuroepithelium and receptor-free epithelium by this time. The vomeronasal organ receives a separate arterial blood supply arising from septal tributaries of the olfactory artery, a branch of the anterior cerebral artery from the earliest stage of development. Blood from the vomeronasal complex is collected in the vomeronasal vein lying in the longitudinal furrow next to the receptor-free epithelium. The typical vascular pattern of the vomeronasal organ is established by the eighteenth day of gestation. At this time, the first capillary loops appear within the neuroepithelium and the vomeronasal vein can already be seen to extend along the long axis of the organ.

Renal vascular changes in hydronephrosis in rats: scanning electron microscopy of resin casts of glomerular capillaries.

Vascular changes in the hydronephrotic glomeruli of rats were examined using corrosion casts, India ink injection and histological methods. In severe cases, the diameter of the glomerular capillary conglomerations decreased to 40-90 microns as a result of glomerular atrophy. The capillaries of these glomeruli also had reduced diameters, and were strangulated and torn off to varying degrees. The intact capillary network of the renal medulla was destroyed and the normal alignment of capillaries parallel to the tubules was lost. These morphological characteristics may account in part for the difficulty in glomerular capillary blood circulation.

The distribution and structure of the lymphatic system in dog atrioventricular valves.

A morphological study of the lymphatic system in atrioventricular (AV) valves of adult dogs and puppies was carried out using a series of techniques including India ink injection, a hydrogen peroxide technique, light microscopy, and electron microscopy. Lymphatic capillaries were found in all cusps of the AV valves, and were shown to extend delicate networks in the subendocardium of the atrial side of the valves. The extent of their development varied among cusps, being most prominent in the anterior cusp of the mitral valve. Marked differences were noted in the intravalvar distribution between lymphatic capillaries and blood microvessels. The ultrastructures of the lymphatic capillaries in the AV valves were also demonstrated.

Effect of neural crest ablation on development of the heart and arch arteries in the chick.

Mesenchymal derivatives of the neural crest contribute to the connective tissues and blood vessels of the pharyngeal arches, and participate in the septation of the outflow tract of the heart. The present study was designed to determine the nature and timing of alterations in the development of the heart and arch arteries subsequent to diminished neural crest contributions. The neural crest contributing to the three caudalmost pharyngeal arches was ablated bilaterally in chick embryos and compared with sham or unoperated controls. Heart development was studied by scanning electron microscopy. Arch artery development was studied microscopically after intravascular injection of India ink and clearing of the specimen. Neural crest ablation caused morphological changes in most hearts. Hearts in experimental animals commonly were elongate and were subject to inappropriate development of ventricular and atrial areas. A surgical effect delayed the disappearance of arch arteries one and two, and removal of neural crest produced an additional delay. Neural crest ablation caused failure of arch arteries three, four (right), and six to develop to the proper size in some animals. Survival of those whose sixth arch arteries achieved the proper size caused group measurements to reach normal values again by stage 32. Closure of arch arteries in some animals and maintenance in others produced greater variability in experimental animals than in controls. It is significant that heart morphology was altered before septation of the outflow tract normally occurs. This indicates at the least that another factor, such as altered blood flow, contributes to the abnormal development. Altered flow may result from changes in pharyngeal arch mesenchyme and arch artery endothelium.