India Ink Injection Research Articles

Uptake of India ink particles and latex beads by corneal fibroblasts

The fate of India ink particles and polystyrene latex beads injected into the corneal stroma of rabbits was studied by the naked eye, light microscopy, and electron microscopy. All the injected ink particles or latex beads were unchanged in shape, size, and number for at least 6 months. India ink particles and latex beads were endocytosed by the corneal fibroblasts within 3-4 days after injection. Numerous ink particles were packed into vacuoles, 0.5-10 micron in diameter, which occupy a large volume of the cytoplasm of the cell body and processes of fibroblasts in and near the injected area. Each latex bead, 0.72 micron in diameter, is usually enclosed in one vesicle, and a large number of vesicles are distributed throughout the cytoplasm. In corneal tissue removed 10 min after injection of India ink and cultured for 3 or 7 days, uptake of many ink particles by the fibroblasts was seen. By this experiment, the contribution of the blood-derived cells was completely excluded, and it is more distinctly shown that the corneal fibroblast has a strong endocytotic activity. The uptake and long-term storage of ink particles and latex beads by the corneal fibroblast are reactions that protect the organ without inflammation from the injury and harm by non-toxic foreign materials.

The Osteocutaneous Free Scapular Flap for One-Stage Mandible Reconstruction

At the recent spring meeting of the American Academy of Facial Plastic and Reconstructive Surgery in Denver, Shan R. Baker, MD, and Michael J. Sullivan, MD, of the University of Michigan, Ann Arbor, presented their experience using revascularized bone harvested from the scapula for mandibular reconstruction. Unlike some other donor sites, advantages of the free scapular flap include its long vascular pedicle and relative thinness. As much as 14.5 cm of bone can be harvested in male patients, and 10.5 cm can be harvested in female patients. Baker also described cadaver studies in which India ink injections of the subscapular artery showed a wide area of skin profusion, as well as staining of the periosteum and cancellous bone of the scapula. Since it has its own vascular pedicle, the skin paddle can be rotated through a wide arc relative to the axis of the bone graft. Baker presented the experience

The healing of femoral neck fractures in dogs

This study evaluates healing fractures of the femoral neck in dogs by using standard radiography, intraosseous venography, India ink staining and histology. The 45 animals were divided into three groups, one of which was not treated, but in the other two the fracture was fixed with a screw or two Kirschner wires. Our results suggest that, when intraosseous venography was positive India ink injection was also positive, and the late phase of fracture healing was reached.

The effect of long-term irradiation with fluorescent light on the rat fundus.

In the rat, continuous exposure to so-called "cold light" of 800 lux leads to a nearly complete loss of retinal photoreceptors, which occurs within 4 weeks. This process is followed by vascularization of the retinal pigment epithelium (RPE). The origin, extent, and expansion of such proliferations within 6 months after the onset of irradiation were evaluated by means of light microscopy, transmission electron microscopy, scanning electron microscopy, corrosion cast preparation, india ink injection, and trypsin digest preparation. The vessels within the RPE were found to be fenestrated and to form a network with the vessels of the neurosensory retina. Invasion of Bruch's membrane and formation of chorioretinal anastomoses were not observed. The occurrence of vessels within the RPE coincided with a massive regression of capillaries in the neurosensory retina.

Structure and function of the melano-macrophage centres of the goldfish Carassius auratus

We have studied the melano-macrophage centres (MMCs) of Carassius auratus as well as the changes they undergo in different experimental conditions. The MMCs of the spleen contain brown and yellow pigments, but dark pigments abound in those of the kidney. They occur in association with splenic ellipsoids and scattered throughout the renal parenchyma. Histochemical tests demonstrated small amounts of lipids and basic proteins, abundant neutral mucopolisaccharids and differences between the spleen MMCs and those of the kidney, specially regarding the presence or absence of haemosiderins and melanins. Electron microscopy shows them to consist of phagocytes containig degenerated debris, sometimes surrounded by a discontinuous layer of flat reticular cells. The MMCs of the spleen and kidney trap carbon material 48 hours after injection of Indian ink. Primary and -more so- secondary immunization with SRBC produces a net increase in number and size of MMCs. Treatment with phenylhydrazine produces a notable and fast increase of the MMCs in both organs. After 5 days the MMCs degenerated and erythroblasts restored the erythroid population. Apparently, MMCs represent non-specific means of phagocytosis related to different physiological processes.

The effect of the thromboxane synthetase inhibitor Dazmegrel (UK-38,485) on wound healing, dermal ink perfusion and skin blood flow measurements in deep partial thickness burns

A guinea-pig model was used to evaluate the effect of Dazmegrel (UK-38,485), a new highly selective inhibitor of thromboxane synthetase, on burn wound healing. Dazmegrel had a beneficial effect on wound healing when given systemically at a dosage of 3·4 mg kg −1day −1 but higher doses had no effect. There was no change in dermal perfusion measured by India ink injection or by 133Xe injection in any of the groups receiving parenteral Dazmegrel. Neither was the number of hair follicles in the healing burn wound significantly different between the treated and control animals. When Dazmegrel was applied topically, it inhibited wound healing. These findings are consistent with previous reports from different laboratories that prostaglandin inhibitors impair healing of the burn wound when applied topically but improve wound healing when given parenterally. The present study further suggested that the improvement in wound healing in this model was not mediated by improved dermal perfusion.

Localized vascular regression during limb morphogenesis in the chicken embryo: II. Morphological changes in the vasculature.

The regression of blood vessels in the distal wing bud of chicken embryos from stages 19 to 31 was examined by light and electron microscopy. The vessels were double-labelled by an injection of Monastral blue B (MB) to label the regressing endothelial cells, followed 6-48 hours later with an injection of India ink which marked the lumens of patent vessels. Prior to stage 26 the vessels contained only India ink since the endothelial cells were not phagocytic at this stage. Vessels at stage 26 or later were often double-labelled, with MB sequestered in the endothelial cell cytoplasm and India ink in the vessel lumens. After stage 27 cells not associated with lumens, but labelled with MB, were observed in areas undergoing vascular regression. Ultrastructural changes in the endothelial cells as the vessels regressed included formation of luminal and abluminal processes, long complex junctions, and vacuoles containing MB. In many involuting vessels the endothelial cells appeared normal even though the lumens were collapsed. Occasionally, isolated pyknotic cells were observed in regions that had been previously vascularized. At stage 31 cells in the developing cartilage had vacuoles containing MB. Our study suggests that blood vessels may disappear from the prechondrogenic zone of the distal wing bud by several mechanisms. These could include a type of cell death that does not elicit a cellular infiltrate, migration of the endothelial cells away from vascularized regions, and/or transdifferentiation into cells that resembled chondrocytes.

Vascular reorganization and return of rigidity in fracture healing.

The present study addresses the relationship between vascular reorganization and the biomechanical changes occurring during canine long bone fracture healing. A middiaphyseal osteotomy of the radius was performed, and angiographic studies were carried out using India ink injections from 2 to 12 weeks postosteotomy. The radii were harvested, subjected to nondestructive four-point bend testing on an Instron, and then sectioned and cleared according to the Spaltholtz technique. The observed microangiographic patterns were correlated with the calculated rigidity values. Quantitative bone blood flow measurements around the osteotomy were performed on a different group of animals, using the radiotracer microsphere technique, at 3, 6, and 9 weeks postosteotomy to correlate with our qualitative microangiographic results. We demonstrated a direct and predictable relationship between the phase of vascular reorganization and the rigidity. This correlation was present even in our delayed union specimens.

Pedicled scapular apophysis transplantation: Growing grafts studied in dogs

Twenty-eight immature dogs, divided into three groups, have been studied to determine the possibilities of pedicled epiphyseal transplantation from a rarely used donor site area, the scapula. One group of 8 immature dogs were studied anatomically using India ink and Batson's compound injection methods. We identified the vascular pattern of the diaphyseal portion of the graft, consisting of the lateral crest or border of the scapula. In two groups of 10 immature dogs, this area was used as a pedicled island graft to rebuild the semiresected or totally resected proximal humerus. There were marked differences between these two groups on the basis of morphologic, histologic, histochemical and electron microscopic aspects of the samples. Preservation of normal growth appeared to be directly related to the compressive forces applied to the graft. Preservation of the cartilage was also related to the biomechanical situation; fibrocartilage and signs of arthrosis were noted 1 year after the operation in several animals whose humeri had been fully subjected to normal stresses. Both the diaphyseal and epiphyseal areas had increased one- to fivefold in volume, but longitudinal growth was markedly deficient.

Effect of respiratory motion on pulmonary activity determinations by positron tomography in dogs.

The effect of respiratory motion on pulmonary activity determinations by positron emission tomography (PET) was studied in dogs with experimentally created pulmonary emboli (PE). The location of the PE was evaluated by planar 99mTc lung imaging to determine the appropriate sites for transaxial PET scans. PET scans of the lung then were obtained after i.v. injection of 68Ga-labeled microspheres. PET scans were acquired during slow (15 breaths/min) and fast (30 breaths/min) breathing with the same minute ventilation and then postmortem. Lung perfusion patterns were documented by i.v. injection of India ink before sacrifice. Cross sections of the excised lungs were made at the same levels as the PET scans, and eight sections containing 14 perfusion defects were analyzed. The scans obtained during slow breathing consistently showed edge blurring and demonstrated defects less well than scans obtained during fast breathing or postmortem. The normal-to-defect activity ratios during fast breathing and on the postmortem studies were similar and approximately 17% higher (P less than .01) than in scans obtained in the same animals during slow breathing. The results demonstrate the need for motion correction during quantitative analysis of regional lung activity by positron tomography, and suggest that high-frequency respiration at small tidal volumes may be one means for obtaining this correction.

Vascular regression during the formation of the free digits in the avian limb bud: a comparative study in chick and duck embryos

The pattern and structure of the blood vessels of the interdigital spaces of the leg bud have been studied by means of Indian ink injections and transmission electron microscopy in the chick and duck embryos. The results show that in the chick the interdigital necrotic process responsible for the freeing of the digits is followed by regression of the blood vessels. In the webbed foot of the duck, the interdigital necrotic processes are not followed by vascular regression. Transmission electron microscopic studies show that both in the chick and in the duck, interdigital blood vessels are immature structures lacking basal lamina. Dead cells of presumably endothelial origin were detected in the lumen of the regressing blood vessels of the chick but not in the duck. However, the intensity of this cell death process does not appear to be high enough to account by itself for the disappearance of the interdigital blood vessels. The possible relationships between interdigital mesenchymal cell death and vascular regression are discussed.

Vasculogenesis in the chick embryo optic tectum.

The initial formation and further development of the intraneural blood vessel network in the tectum opticum of the chick from the 4th to the 14th incubation day have been analyzed and some quantitative data morphometrically recorded. Vessels have been filled by intracardial injection of India ink in vivo. As inferred from our previous investigations on the vasculogenesis of several districts of the central and peripheral nervous system in the chick embryo, also in the developing optic tectum growth and distribution pattern of the vessels seem to unfold step by step under the local influence of earlier occurring morpho-histogenetic processes of the corresponding neural substratum.

Communicatory routes connecting the middle ear, the inner ear and the subarachnoid space via perineural space.

Twenty-one rabbits received injection of Indian ink into the stylomastoid foramen. Observation of the serial sections of their temporal bones revealed Indian ink infiltration into the subarachnoid space, along the facial nerve, the auricular branch of the vagus nerve and the tympanic nerve. Along the acoustic nerve, the infiltration was found as far as the fundus of the internal auditory meatus. In some cases, however, Indian ink reached the subarachnoid space and infiltrated into the scala tympani, scala vestibuli and around the saccule by way of the cochlear aqueduct. These routes are suspected to be a course of spreading infection or tumor cells.

The salutary effects of the bed on the survival of experimental flaps.

The present study indicates that the distal, random segment of an axial pattern experimental flap can be partially salvaged by employing local pressure, which provides favorable contact with the bed. This portion of the flap could be considered as a composite skin graft. Serial India ink injections demonstrated that its physiological take resembles that of a split-thickness graft, dominantly depending on the blood vessels emerging from its bed, and to a lesser extent on those provided by its proximal segment and adjacent skin edges. Increasing the topical flap humidity hindered, rather than improved, its survival.

Homografts of the Tympanic Membrane With Malleus; Histological Study In Cat

The present experiments on cats were performed to explore the process of survival and take of transplanted homografts of tympanic membrane with malleus, the homografts being respectively preserved in 4% and 0.5% formalin (pH 7.0) solutions, and to explore the reaction of middle ear mucosa of the recipient. The present study also included gross and histopathological examinations of the eardrum and observation of fibers of the lamina propria of the transplanted tympanic membrane by means of polarizing microscopy and of vascularization by intravascular India ink injection. The experiments conducted using a total of 96 cats yielded results leading to the following conclusions. 1) After six months the transplanted tympanic membrane showed nearly normal histologic features, with a fibrous layer between the lining epithelial and thin mucosal layers. The middle ear mucosa also became thinner and was covered with ciliated epithelium at this stage. 2) As regards vascular distribution, most vessels entered from the peripheral regions to become distributed all over the eardrum with mutual anastomoses. 3) Fibrous tissues of the lamina propria of the pars tensa of the transplanted eardrum remained intact over the six month period. 4) The transplanted malleus was neither destroyed not absorbed, and proved to survive well with active vascularization. 5) The microscopic changes observed in the middle ear mucous membrane of the recipient showed non-specific reactions to Gelfoam. The grafts were not subjected to immunological rejection. Homograft thus offer the best grafting material, but seemed to show poor resistance to infection.

Vascularization in infantile brain tumors induced by human adenovirus type 12 in rats.

Early vascular changes in the undifferentiated infantile brain tumors induced by human adenovirus type 12 (Ad 12) in rats were studied using the India ink injection method, scanning electron microscopy (SEM) of vascular corrosion casts, transmission electron microscopy, and the peroxidase anti peroxidase method for glial fibrillary acidic protein detection. The process of vascularization of solid type tumors was divided into five phases: Stage O, no remarkable changes; Stage I, dilatation of vessels; Stage II, formation of vascular network; Stage III, fine branching in the vascular network; and Stage IV, necrosis. SEM of vascular replica in Stage III suggested compression of the vessels by proliferating tumor cells. In the tapetum region, Stage I was recognized in solid type tumors 100 to 230 (m = 180) micrometer in diameter, while in the olfactory lobes and in the anterior horn of lateral ventricle closely adjacent to the vascular gray matter, Stage I occurred in much larger tumors. In the diffuse type tumors, the dilatation of vessels was first noticed when the tumors were about 1600 micrometers in diameter. The solid type tumors mainly developed in the poorly vascular subventricular zone, while the diffuse type ones were usually found deep in the vascular gray matter. The results indicate that vascularization is closely related to the local vascularity and the growth pattern of the microtumors, and that of the solid type tumors induced by Ad 12 may be a model of vascular changes in infantile brain tumors which mostly develop and grow rapidly in the subventricular zone which have poor vascular development.

The india ink injection technique to assess the depth of experimental burn wounds.

A detailed description of the India ink injection technique for assessment of experimental burn wound depth is presented with emphasis upon special technical manipulations. Assessment of the burn depth is mandatory for the establishment of a burn wound model as well as to reproduce consistent data among different investigators.

In vivo effects of lipopolysaccharide on lymphoid and non-lymphoid cells in the mouse spleen. Reduction of T-lymphocytes and phagocytosis in the inner parts of the periarteriolar lymphocyte sheath.

In the present study the effects of lipopolysaccharide (LPS) on the cellular composition and phagocytosis of India ink in the inner parts of the periarteriolar lymphocyte sheaths (PALS) are described. Staining for B-, T-lymphocytes, and reticulin fibers in the spleen of normal and LPS-injected mice shows that the B-dependent follicular area is increased in size after LPS administration. However, the number of T-lymphocytes in the inner PALS is reduced markedly and a relatively high number of B-lymphocytes can be found in this area. The significance of this phenomenon is discussed. In untreated mouse spleen, carbon particles become localized in strongly acid-phosphatase (AP)-positive macrophages of the red pulp, marginal zone and white pulp 24 h after an intravenous injection of India ink. All these macrophages contain numerous carbon particles. After LPS pretreatment, the phagocytosis of carbon particles in the inner PALS is dramatically diminished, although many strongly AP-positive macrophages can be found in this area. The phagocytosis of carbon particles in the other compartments of the spleen did not change. It appears that injection of 2 micrograms LPS or more is sufficient to induce this phenomenon which is most significant when LPS is injected 24 or 48 h before exposure to India ink.

Proliferative patterns of lymphocytes in lymph nodes during tumour development: involvement of T and B cell areas.

DNA-synthetizing lymphocytes were identified in the lymph nodes regional and more distal to the site of developing P-815 tumours by incorporation of [3H]-thymidine followed by autoradiography of lymph node sections. It appeared that not only T but also B cell areas of draining and to a lesser extent of distal lymph nodes were stimulated by the growing tumour. This result was unexpected since neither humoral nor tumour cell-bound antibody could be identified so far as a functional correlate of B cell stimulation. In general the proliferative response of lymphocytes followed a biphasic pattern with an early peak of reactivity on days 2-3 and a second peak around day 12-15 after tumour cell inoculation. In the draining (axillary) lymph node the second peak of reactivity was suppressed, possibly as a consequence of metastatic tumour cells in this node when tumour cells were inoculated in the flank. The pattern of lymphocyte stimulation revealed larger individual variations after tumour cell inoculation in the flank than the foot pad. These results were associated with a slower and less regular drainage of carbon particles from the flank to the axillary and exceptionally the brachial lymph node than from the foot pad to the popliteal node after injection of India ink.

Demonstration of M cells in the specialized follicle-associated epithelium overlying isolated lymphoid follicles in the gut.

Within the epithelium that overlies the dome regions of Peyer's patches, exist specialized surface epithelial cells (M) which function to take up macromolecules from the gut lumen. These cells may be of great importance in processing antigenic material in the gut. The predominant lymphoid structures of the small intestine are isolated lymphoid follicles, by virtue of their frequency. These follicles are difficult to study because they are not grossly visible. In the present study, three guinea pigs drank India ink mixed into their water for 1, 3, and 5 months. Two hours prior to sacrifice, animals were given an intraintestinal injection of ferritin or India ink. Using a hand lens, the Peyer's patches and isolated follicles were clearly identified among the villi of the intestine. Light microscopy revealed ink in the surface epithelium covering the isolated follicles and within the substance of the follicles. Transmission electron microscopy demonstrated M cells over isolated follicles and Peyer's patches. These cells had lighter staining cytoplasm, while the mitochrondria stained darker with prominent cristae, and the microvilli were shorter. Therefore, M cells do exist within isolated follicles and structurally appear the same as those found in Peyer's patches. This implicates the isolated follicles in the overall antigen processing role of gut-associated lymphoid tissues. The present method facilitates identification of isolated lymphoid follicles which will allow functional studies to be performed on these structures.