Do we really need cadavers anymore to learn anatomy in undergraduate medicine?

With the availability of numerous adjuncts or alternatives to learning anatomy other than cadavers (medical imaging, models, body painting, interactive media, virtual reality) and the costs of maintaining cadaver laboratories, it was considered timely to have a mature debate about the need for cadavers in the teaching of undergraduate medicine. This may be particularly pertinent given the exponential growth in medical knowledge in other disciplines, which gives them valid justification for time in already busy medical curricula. In this symposium, the pros and cons of cadaver use in modern medical curricula were debated and audience participation encouraged.

The biceps brachii muscle and its distal insertion: observations of surgical and evolutionary relevance.

BACKGROUND: A sound understanding of the anatomy of the biceps brachii and possible anatomical variants is necessary to manage distal biceps injury. The present study was performed to define the anatomy of the biceps brachii with particular focus on the conformation of the distal biceps tendon, and its relationship of the two heads of the biceps brachii. METHODS: Twenty cadaver specimens were dissected and both qualitative and quantitative observations were made of a series of features relating to the biceps muscle and its tendon. RESULTS AND CONCLUSION: The investigation revealed anatomical variations including supernumerary heads (20%) and 'fusion' of the muscle proximal to tendon formation and a spiralling arrangement of the tendon in its approach to the radial tuberosity. The data from the present study was reviewed in the context of previous studies on the anatomy of this muscle and speculation on the evolutionary basis of the variations and their clinical implications are discussed.

Resident tissue macrophages within the normal rat iris lack immunosuppressive activity and are effective antigen-presenting cells.

Despite extensive study of the numerous immunoregulatory mechanisms that contribute to the 'immune-privileged'nature of the anterior chamber (AC) of the eye, little is known of the functional nature of antigen-presenting cells (APC) present in the tissues adjoining the AC. In the present study, we have compared the antigen-presenting capacity of dendritic cells (DC) and macrophages isolated from the normal rat iris. Whereas iris DC exhibited a potent ability to stimulate resting allogeneic T cells in MLR cultures (an in-vitro correlate of the ability to induce primary T cell responses), resident iris macrophages displayed negligible MLR-stimulatory capacity. Significantly, iris macrophages could efficiently elicit proliferation of primed antigen-specific T cells (an in-vitro correlate of the ability to act as local APC in secondary responses). This antigen-presenting activity was approximately half that of fully 'mature' iris DC and considerably greater than that of freshly isolated iris DC. A key contributor to the effectiveness of resident iris macrophage antigen presentation was considered to be the absence of lymphocytostatic control of T cell proliferation exerted by these cells. The results indicate dichotomous but complementary roles for DC (immune surveillance) and macrophages (local antigen presentation in secondary responses) in this tissue.

Analysis of the cellular infiltrate in the iris during experimental autoimmune encephalomyelitis.

PURPOSE: Previous studies have shown that experimental autoimmune encephalomyelitis (EAE) and anterior uveitis (AU) develop in Lewis rats immunized with myelin basic protein (MBP). The purpose of this study was to characterize the dynamics, distribution, and phenotype of infiltrating cells in the iris during EAE-associated AU. METHODS: Lewis rats were immunized with MBP emulsified in complete Freund's adjuvant (CFA) or with CFA alone. Cellular infiltration of the iris was analyzed at various time points by immunohistochemistry of wholemounts, flow cytometry, and immunoelectron microscopy, by using monoclonal antibodies specific for monocytes/macrophages (ED1), T lymphocytes (R73, W3.25, OX8), T-cell activation markers (OX39, OX40), granulocytes (HIS48), major histocompatibility complex (MHC) class II (OX6), and neurofilament (2H3). RESULTS: MBP-immunized rats showed development of characteristic monophasic EAE, followed, after resolution of paralysis, by mild self-limited AU. Initially, focal infiltrates of round MHC class II(+) and ED1(+) cells were found in the iris. During the course of AU, the midiris became massively infiltrated with ED1(+) monocytes-macrophages, R73(+) T cells, granulocytes (HIS48(+)), and MHC class II(+) cells. The influx of T cells consisted of CD4(+) and CD8(+) cells, of which only a small fraction (<14 and 11%, respectively) expressed activation markers. The infiltrating cells accumulated in proximity to myelinated and nonmyelinated nerve bundles and in the vicinity of blood vessels in the iris. No evidence was found for demyelination or nerve degradation. Neither EAE nor AU developed in CFA-treated control rats. CONCLUSIONS: These data show that EAE-associated AU is characterized by a transient mixed cellular infiltrate consisting of monocytes-macrophages, granulocytes, and CD4 and CD8 T cells. The preferential accumulation of inflammatory cells in the vicinity of nerve fibers suggests that AU in this model may result from autoreactivity to nerve antigens.

Optimal methods for preparation and immunostaining of iris, ciliary body, and choroidal wholemounts.

PURPOSE: Investigations into the biology of resident and infiltrating immune cells in the uveal tract of the rodent eye have been greatly aided by the use of tissue wholemount methods. These methods offer a number of advantages over conventional histological and frozen section techniques. The purpose of this article is to provide a detailed step by step guide to aid others who may wish to use this method. METHODS: A detailed description of whole-body perfusion fixation, dissection and isolation of the iris-ciliary body from the anterior segment and the choroid from the posterior segment is provided. In addition, the techniques used to handle whole tissue pieces during single and double immunohistochemical staining protocols, as well as the staining protocols themselves, are described. RESULTS: In refining the techniques described, the author has catalogued a number of frequent problems which compromise immunohistochemical staining results. A troubleshooting guide aimed to help identify the cause of common problems and with some suggested remedies is provided. CONCLUSIONS: Although tissue wholemounts are frequently used in retinal research, a similar approach to investigating the components of the uveal tract has only recently been applied. The methods described in this article will provide sufficient detail for other investigators to obtain maximum benefit from this alternative approach and provide an additional technique to assist in their investigations of ocular immunobiology.

Environmental scanning electron microscopic study of macrophages associated with the tunica vasculosa lentis in the developing rat eye.

AIMS: To demonstrate the value of environmental scanning electron microscopy (ESEM) when used in combination with immunogold/silver enhancement methods as a valuable tool in ocular research, and to determine the phenotype of macrophages associated with the tunica vasculosa lentis while maintaining a topographical view of the lens surface. METHODS: Prenatal and postnatal rat eyes were investigated by conventional scanning electron microscopy and ESEM. In the latter case tissues were prestained with a panel of antileucocyte monoclonal antibodies and visualised with colloidal gold conjugated secondary antibody followed by silver enhancement. RESULTS: The preliminary data demonstrate that ED1(+) ED2(+) macrophages occur in large numbers around the lens and are associated with sectors of both normal vessels and those undergoing regression. CONCLUSION: The present study demonstrates that ESEM is an ideal way to combine scanning electron microscopy with immunohistochemistry and is therefore likely to have multiple other applications in ocular research.

Subretinal macrophages in the developing eye of eutherian mammals and marsupials.

Blood-borne mononuclear cells invade the developing retina via the hyaloid vasculature at the optic nerve head. Following removal of apoptotic cell debris they give rise to the network of resident microglia. The population of cells recently described in the peripheral subretinal space of developing human eyes may represent a further population of macrophages destined to become microglia. The aim of the present study was to confirm the presence of subretinal macrophages in the developing eye in other mammalian species and perform preliminary immunophenotypic analysis in rat tissues. The range of species chosen included eutherian mammals (rat and rabbit) and marsupials (wallaby and opossum). Ocular tissues from a range of developmental stages were studied by scanning electron microscopy and transmission electron microscopy. Distinctive networks of dendriform and pleomorphic macrophages were observed by scanning electron microscopy in the peripheral subretinal space of D2 rabbits, newborn and D2 rats and D75 wallaby. Transmission electron microscopic studies of D2 rabbit, newborn and D2 rat and all ages of North American opossum revealed cells with the ultrastructural features of macrophages in the peripheral subretinal space, cilio-retinal junction and between ciliary epithelial cells. Preliminary immunoperoxidase studies using a panel of anti-leukocyte monoclonal antibodies on frozen sections of rat ocular tissues (newborn, D2 and D4) revealed ED1(+) Ox42(+) ED2(+) but Ox6(-) cells in the peripheral subretinal space, peripheral retina and ciliary body epithelia. The data confirms that subretinal macrophages are a feature of the developing eye in a broad range of mammalian species and immunophenotypic evidence leads the author to postulate that these cells arise from the ciliary body vasculature and may migrate into peripheral neural retina and mature into resident microglia.

Dendritic cells and macrophages in the uveal tract of the normal mouse eye.

BACKGROUND/AIMS: Dendritic cells (DC) and macrophages are components of the immune cell populations in the uveal tract whose density, distribution, turnover, and function may play a role in the maintenance of immunological homeostasis in the eye. Little is known of these cells in the mouse eye despite this being the predominant experimental model in many studies of ocular immune responses and immunoinflammatory mediated eye diseases. The aim of the present study was to obtain further immunophenotypic data on resident tissue macrophages and DC populations in the mouse uveal tract. METHODS: Pieces of iris, ciliary body, and choroid dissected from perfusion fixed BALB/c mice were incubated whole in a variety of anti-macrophage and DC monoclonal antibodies (mAbs). Labelled cells were visualised using either single or double immunoperoxidase techniques. RESULTS: Quantitative analysis and double immunolabelling revealed that 80% of F4/80(+) cells (a mAb that recognises both DC and macrophages) in the iris are macrophages (SER4(+)). The iris contained a network of Ia+ cells (412 (SD 130) cells/mm2) of which two thirds appear to be DC. A similar pattern was observed in the ciliary body and choroid. Only a few DC in the uveal tract were very weakly reactive for mAbs which recognise B7-1 (CD80), B7-2 (CD86), beta2 integrin (mAb N418), and multivesicular bodies associated with antigen presentation (mAb M342). CONCLUSIONS: The present study reveals that the mouse uveal tract, like the rat, contains rich networks of DC and resident tissue macrophages. The networks of resident tissue macrophages in the mouse uveal tract closely resemble similar networks in non-ocular tissues. The phenotype of uveal tract DC suggests they are in the "immature" phase of their life cycle, similar to Langerhans cells of the skin, thus implying their role in situ within the eye is antigen capture and not antigen presentation.

Distribution and phenotype of dendritic cells and resident tissue macrophages in the dura mater, leptomeninges, and choroid plexus of the rat brain as demonstrated in wholemount preparations.

Dendritic cells (DC) are regarded as the 'sentinels' of the immune system. They play a crucial role in surveillance of peripheral tissues, trapping antigens encountered there, and migrating via the lymphatics to lymphoid organs where they interact with naive T cells thus generating antigen-specific primary immune responses. Until now it has been assumed DC are largely absent from the brain, meninges, and the choroid plexus within the ventricles. Such a situation was thought to partly explain the 'immune privileged' nature of the central nervous system (CNS). The present study of normal rat tissues using single and double immunohistochemistry reveals for the first time that extensive networks of major histocompatability (MHC) class II+/OX62+ DC are widely distributed in sites which may potentially encounter CNS antigens. These sites included the dura mater, leptomeninges, and the choroid plexus. These putative DC were negative when stained with the anti-resident tissue macrophage monoclonal antibody ED2. In addition to the rich networks of DC, dense populations of resident tissue macrophages (ED2+ and ED1+) were also demonstrated in the dura mater, leptomeninges and to a lesser extent in the choroid plexus. The presence of rich networks of DC and macrophages in the vascular and supporting tissues of the brain may play an important role in inflammatory and immune-mediated disorders affecting the CNS, including auto-immune demyelinating diseases such as multiple sclerosis.

Major histocompatibility complex (MHC) class II--positive dendritic cells in the rat iris. In situ development from MHC class II-negative precursors.

PURPOSE: To examine the postnatal development of major histocompatibility complex (MHC) class II-positive dendritic cells (DC) in the iris of the normal rat eye. METHODS: Single- and double-color immunomorphologic studies were performed on whole mounts prepared from rat iris taken at selected postnatal ages (2 to 3 days to 78 weeks). Immunopositive cells were enumerated, using a quantitative light microscope, and MHC class II expression on individual cells was assessed by microdensitometric analysis. RESULTS: Major histocompatibility class II-positive DCs in the iris developed in an age-dependent manner and reached adult-equivalent density and structure at approximately 10 weeks of age, considerably later than previously described in other DC populations in the rat. In contrast, the anti-rat DC monoclonal antibody OX62 revealed a population of cells present at adult-equivalent levels as early as 3 weeks after birth. Dual-color immunostaining and microdensitometric analysis demonstrated that during postnatal growth, development of the network of MHC class II-positive DCs was a consequence of the progressive increase in expression of MHC class II antigen by OX62-positive cells. CONCLUSIONS: During postnatal growth, the DC population of the iris develops initially as an OX62-positive-MHC class II-negative population, which then develops increasing MHC class II expression in situ and finally resembles classic DC populations in other tissue sites. Maturation of the iris DC population is temporally delayed compared with time to maturation in other tissue sites in the rat.

Cellular localisation and dynamics of nitric oxide synthase expression in the rat anterior segment during endotoxin-induced uveitis.

The present study examined the temporal pattern and cellular localisation of nitric oxide synthase in Endotoxin-Induced Uveitis (EIU). Lewis rats (n=40) received a single footpad injection of 200 microg of bacterial lipopolysaccharide. Animals were killed at 0, 2, 4, 6, 12, 24, 48 and 72 hr after injection and ocular tissues prepared as iris-ciliary body wholemounts or frozen sections of the anterior segment. The expression of constitutive nitric oxide synthase (cNOS) and inducible nitric oxide synthase (iNOS) was investigated at all time points by immunohistochemistry. A further group of animals (n=6) were killed at the peak of the disease (12 hr) and the cellular co-localisation of iNOS on resident and infiltrating immune cells was investigated by double immunohistochemistry utilising the biotinylated monoclonal antibodies ED1, ED2 and Ox6. Expression of cNOS on iris vessels did not alter during the course of EIU. Quantitative analysis of iris-ciliary body wholemounts revealed the first evidence of iNOS+ at 2 hr which increased dramatically at 4 and 6 hr with a peak at 12 hr. The expression of iNOS in the early phase of the disease (2-6 hr) was associated with small round marginating and newly extravasated cells that on morphological criteria were most likely neutrophils and monocytes. At 12 hr, cells of more mixed morphologies began to express iNOS and double labelling revealed 70% of these cells were also ED1(+) (a lysosomal antigen present in monocytes/macrophages and dendritic cells), 52% were Ox6(+) (MHC class II) (dendritic cells, activated macrophages and some T-cells) and 19% were ED2(+) (pan-specific resident tissue macrophages). Expressed in an alternative manner, 10% of the total ED1(+) cell population, 11% of the ED2(+) cells and 44% of Ox6(+) cells co-expressed iNOS. Expression of iNOS decreased significantly by 24 hr to near baseline levels and was absent by 48 and 72 hr. Within the ciliary processes iNOS+ dendriform cells were noted at 6 hr and accumulations of many small round iNOS+ cells were present at 12 hr. The ciliary epithelium did not at any time express iNOS at the protein level detectable by immunohistochemistry. The results of this study suggest that iNOS expression early in EIU is associated with infiltrating or newly recruited neutrophils and monocytes/macrophages in the iris whereas later in the disease resident tissue macrophages and MHC class II+ cells (activated macrophages and putative dendritic cells) in the iris and ciliary body may synthesise nitric oxide. The role of this late phase of nitric oxide synthesis may include lymphocytostasis and immunosuppression as proposed in other tissue sites. The outcome of the present study may help in planning therapeutic strategies using NOS inhibitors.

Macrophages and dendritic cells in normal and regenerating murine skeletal muscle.

Mononuclear phagocytes and MHC class II+ dendritic cells (DC) were identified in frozen sections of skeletal muscle using a panel of pan-specific antimacrophage (MOMA-2, SER-4, Mac-1, F4/80), anti-major histocompatibility complex (MHC) class II (M5/114) and anti-DC (NLDC-145, N418, M342) monoclonal antibodies. Uninjured and regenerating skeletal muscle were investigated in SJL/J and BALB/c mice, strains with known differences in muscle regenerative capacity. Resident tissue macrophages and MHC class II+ DC were present within uninjured mouse muscle. A subpopulation of DC were positive for the pan-DC markers, N418 and M342, and negative for the lymphoid DC marker NLDC-145. Following crush injury, the macrophage population increased by day 2, became marked by day 3, and had decreased by day 6. In contrast, the number of MHC class II+ cells around the injury site increased steadily after injury and remained high at day 6. The numbers of macrophages and DC detected by immunohistochemical staining were consistently higher in SJL/J than BALB/c muscles. This study confirms that macrophages are a significant component of normal murine skeletal muscle and that these cells increase dramatically after injury. Furthermore the data also reveal for the first time that DC are present in normal skeletal muscle and that MHC class II+ cells, including DC, increase after injury. The presence of DC in muscle has important implications for the understanding of the immunobiology of muscle and immune-mediated processes such as the host versus graft responses following muscle transplants and autoimmune diseases affecting this tissue.

The distribution of immune cells in the uveal tract of the normal eye.

Inflammatory and immune-mediated diseases of the eye are not purely the consequence of infiltrating inflammatory cells but may be initiated or propagated by immune cells which are resident or trafficking through the normal eye. The uveal tract in particular is the major site of many such cells, including resident tissue macrophages, dendritic cells and mast cells. This review considers the distribution and location of these and other cells in the iris, ciliary body and choroid in the normal eye. The uveal tract contains rich networks of both resident macrophages and MHC class II+ dendritic cells. The latter appear strategically located to act as sentinels for capturing and sampling blood-borne and intraocular antigens. Large numbers of mast cells are present in the choroid of most species but are virtually absent from the anterior uvea in many laboratory animals; however, the human iris does contain mast cells. Small numbers of what are presumed to be trafficking lymphocytes are present in the uveal tract of normal eyes. There is little data available on the presence or absence of eosinophils. The role of these various cell types in immune homeostasis and ocular inflammation is briefly considered.

Resident and infiltrating cells in the rat iris during the early stages of experimental melanin protein-induced uveitis (EMIU).

Experimental melanin protein-induced uveitis (EMIU) is reported to be a model of anterior uveitis and choroiditis in which the retina is spared. In this study, we chose to compare EMIU with experimental autoimmune uveoretinitis (EAU), a well-recognised model of endogenous posterior uveitis, with regard to the nature and dynamics of the cellular infiltrate in the iris. Female Lewis albino rats were immunised with mixtures of crude retinal extract/complete Freund's adjuvant (CFA) (EAU), phosphate-buffered saline/CFA (controls), or iris melanin/CFA (EMIU) using established protocols. Animals were sacrificed on days 10 and 13 (around disease onset). Following whole body perfusion fixation, irides were dissected from the remainder of the globe. Iris wholemount preparations were then subjected to immunohistochemical analysis in order to investigate both the dynamics of infiltrating leukocytes and the effects of the inflammatory changes on resident immune cells in the iris. The nature of the cellular infiltrate in both EMIU and EAU models was essentially similar, namely there was a rich infiltrate of EDI+ mononuclear cells, Ox42+ neutrophils and T cells. Resident tissue macrophages (ED2+) were slightly below normal densities in the iris of EAU animals and marginally elevated in EMIU animals, MHC class II (Ia) staining, associated in the normal eye with dendritic cells (DC), was considerably elevated in EMIU. It is likely that this was due to both increased DC numbers and an influx of Ia+ exudate macrophages. No striking difference was found in the nature and phenotype of the cellular infiltrate in the iris at the onset of the disease in these two models of uveitis (EAU and EMIU). This suggests that the anterior segment inflammation in both models represents non-specific changes secondary to cytokine release associated with interaction of activated antigen-specific T cells and target antigens, namely retinal photoreceptors in EAU and uveal tract melanin-containing cells in EMIU. Alternatively, it may suggest that antigen-presenting cells resident in the iris and ciliary body in normal eyes have access to ocular antigens on both sides of the blood-ocular barrier and are capable of activating circulating antigen-specific T cells in these models.

Resident and infiltrating immune cells in the uveal tract in the early and late stages of experimental autoimmune uveoretinitis.

PURPOSE: To investigate the dynamics of resident and infiltrating immune cells in the choroid and iris during the early and late stages of experimental autoimmune uveitis (EAU) in Lewis rats. METHODS: Uveoretinitis was induced by footpad injection of crude retinal extract and complete Freund's adjuvant with concurrent intraperitoneal injection of Bordetella pertussis. Five experimental (EAU) and five control animals (adjuvant alone) were studied at days 5, 7, 9, 11 (prodromal stage) and 42 (late stage) after immunization. Five normal animals and five animals injected with B. pertussis alone served as further controls. Immunohistochemical localization of resident macrophages, major histocompatibility complex class II (Ia)+ dendritic cells (DC), infiltrating mononuclear cells, and T cells was performed on wholemounts of isolated choroidal and iris tissue. RESULTS: Double immunolabeling confirmed the presence of distinct networks of macrophages (591 +/- 52 cells/mm2) and DC (746 +/- 38 cells/mm2) in the rat choroid. No marked qualitative and quantitative changes were observed in the density or morphologic appearance of ED2+ resident tissue macrophages in the choroid and iris before clinical onset of ocular disease. On day 11, infiltration of ED1+ monocytes had occurred in the iris but not in the choroid; however, marked infiltration of T cells was evident in both choroid (286 +/- 161 cells/mm2) and iris (196 +/- 72 cells/mm2). The total density of Ia+ cells was significantly elevated in the choroid (1152 +/- 192 cells/mm2) at day 11, and small, round Ia+ cells were two to three times more frequent than normal at both sites. The density of T cells and Ia+ cells remained significantly elevated in the choroid and iris in the late stages of EAU. CONCLUSIONS: These data suggest resident uveal tract macrophages undergo no significant alteration in density in the early stages of EAU and that the earliest site of mononuclear cellular infiltrate in EAU occurs in the iris. The increased total density of Ia+ cells in the choroid on day 11 and the presence of significantly increased numbers of small, round Ia+ cells in the iris and choroid may represent increased trafficking of DC in the eye during uveoretinitis. Furthermore, the raised numbers of Ia+ cells, concurrent with the influx of T cells, suggests Ia+ DC and macrophages may act as local antigen-presenting cells in the induction of uveoretinitis.

Origin and steady-state turnover of major histocompatibility complex class II-positive dendritic cells and resident-tissue macrophages in the iris of the rat eye.

Recent studies have identified distinct but co-existing networks of resident tissue macrophages and MHC class II-positive DC present in tissues bordering the anterior chamber of the eye, a site classically regarded as 'immune-privileged'. As the DC network, present at approximately 500 cells/mm2, accounts for virtually all MHC class II immunostaining in these tissues and possesses potent capacity to stimulate primary allogenic responses in vitro, it is proposed that these cells may play an important role in immune surveillance of the anterior chamber. Tissue macrophage and DC population kinetics in the iris were examined by using X-irradiation exposure to interrupt the steady-state renewal of these cells by haematopoietically derived precursors. MHC class II-positive iris DC exhibited a half-life of approximately 3 days, a rapid turnover rate which closely resembled that of DC present in mucosal epithelia. In contrast, the resident tissue macrophage population displayed a considerably slower turnover (half-life of 10-12 days) comparable to that of epidermal Langerhans cells in the present study. Bone marrow transplantation studies confirmed the haematopoietic origin of the iris DC population. The present study provides the first estimates of the steady-state population kinetics of antigen-presenting cell populations in the iris and has important implications for understanding the role of these cells in immunological homeostasis of the anterior chamber.

Immunomorphologic studies of mast cell heterogeneity, location, and distribution in the rat conjunctiva.

Mast cells are crucial components of immediate and some delayed-type hypersensitivity reactions. They play a pivotal role in allergic conjunctivitis and other immunoinflammatory disorders of the ocular surface, yet little is known of their distribution and heterogeneity in the conjunctiva of potential animal models, such as the rat. In this study, mast cell types were investigated in histologic sections and corneal-conjunctival-lid whole mounts by using toluidine blue, alcian blue-safranin, and immunohistochemical staining methods (anti-rat mast cell proteinase [RMCP] antibodies). Quantitative analyses were performed on corneal-conjunctival-lid whole mounts by using the optical dissector procedure to obtain the density of mast cells per unit volume in different regions of the conjunctiva. Single and double immunohistochemical analyses revealed that the mast cells in the conjunctiva of the limbus, fornices, and lid margin were strongly RMCP I+, suggesting that they were of the connective tissue phenotype. Mast cells containing the mucosal mast cell proteinase RMCP II were not present in the normal conjunctiva. Histochemical analysis revealed that the maturity of the connective tissue mast cells, as assessed by the presence or absence of safranin (heparin)-positive granules in their cytoplasm varied in different regions. In the lid margin 60% to 78% of the mast cells were solely alcian blue-positive, whereas in the fornices 68% to 78% were safranin-positive. In the limbus the predominant type of mast cell was either safranin-positive or contained mixed granules. Mast cell densities were greatest close to the lid margin (10,000 to 12,000 cells/mm3), followed by the limbus (3400 to 4800 cells/mm3) and were rare in the remainder of the conjunctiva (500 to 1000 cells/mm3), with the exception of the region around the nictitating membrane. This study of rat conjunctival mast cells provides essential baseline data for future studies of the role of mast cells in models of allergic conjunctivitis.

Inhibition of tumor necrosis factor activity minimizes target organ damage in experimental autoimmune uveoretinitis despite quantitatively normal activated T cell traffic to the retina.

Recent studies demonstrated that administration of a p55-tumor necrosis factor (TNF) receptor IgG-fusion protein (TNFR-IgG) prevented the clinical onset of experimental autoimmune encephalomyelitis but did not alter the number or tissue distribution of autoantigen-specific CD4+ effector T cells which trafficked into the central nervous system. To determine whether specific target tissues of autoimmune damage remain intact after TNFR-IgG treatment despite the presence of inflammatory cells within the tissues, we examined rats with experimental autoimmune uveoretinitis (EAU), as in this model, the main target of autoreactive CD4+ T cells, the retinal rod outer segments (ROS), can be examined readily by light microscopy. As judged by direct ophthalmoscopy, the onset of inflammation in the anterior chamber of the eye in EAU following administration of TNFR-IgG was delayed by 6 days compared to untreated controls, but the magnitude of the response was only slightly less than controls. Histological examination of the retinae and direct assessment of retinal inflammation revealed a disproportionate sparing of ROS in the TNFR-IgG-treated animals despite a level of retinal inflammation not substantially less than controls in which ROS damage was marked. Analysis of retinal leukocytes by immunofluorescence microscopy and flow cytometry indicated that approximately equal numbers of CD4+ alpha beta TCR+ lymphocytes were present in treated and control retinae, more than 30% of CD4+ cells in both experimental groups expressed the CD25 or MRC OX40 activation markers and most cells, which would include the CD4+ T lymphocytes, were activated as evidenced by MHC class II expression. Fewer activated macrophages and granulocytes were present in the treated retinae, possibly reflecting the lower level of tissue damage and subsequent accumulation of these inflammatory cells. The results demonstrate directly that a tissue specifically targeted for autoimmune destruction can be protected despite the influx of fully activated CD4+ T cells.

Endotoxin-induced uveitis. Kinetics and phenotype of the inflammatory cell infiltrate and the response of the resident tissue macrophages and dendritic cells in the iris and ciliary body.

PURPOSE: Footpad injection of bacterial lipopolysaccharide (LPS) causes pronounced anterior uveitis in susceptible species and strains. Recent studies using wholemount techniques have demonstrated the presence of rich networks of major histocompatibility complex (MHC) class II-positive dendritic cells (DC) and resident tissue macrophages in the iris and ciliary body. The aim of this investigation was to determine the immunophenotype and dynamics of the inflammatory cell infiltrate during LPS-induced anterior uveitis using the wholemount method and to examine the response of the resident tissue macrophages and DC to an acute inflammatory episode in the anterior segment. METHODS: Female Lewis rats (8 to 12 weeks old, n = 49) received a single footpad injection of 100 micrograms of LPS and were killed at various times up to 6 weeks after injection. The iris-ciliary body complex from each eye was removed intact and subdivided into segments and immunostained using a panel of monoclonal antibodies to a variety of immune cell types. RESULTS: The wholemount method clearly illustrates that during endotoxin-induced uveitis (EIU), the earliest cellular infiltrate includes small, round ED1+ mononuclear cells marginating in the iris vasculature approximately 2 hours after injection. Marginating Ox42+ polymorphonuclear leukocytes were detectable in the iris vessels approximately 4 to 6 hours after injection and were especially numerous in the ciliary body base approximately 24 hours after injection. The overall density of resident tissue macrophages (ED2+) remained largely unchanged in the course of EIU. In contrast, the total number of MHC class II-bearing (Ox6+) cells (putative dendritic cells) increased 30% in the first 6 hours and 200% by 72 hours. During the acute phase of the inflammatory response (up to 24 hours), the proportion of these cells with a dendritiform morphology decreased (93% to 50%). The number of T cells showed a biphasic response peaking at 4 to 6 hours and again at 24 hours (290 cells/mm2); however, their numbers had resumed normal low density (4 cells/mm2 to 25 cells/mm2) by 6 weeks. CONCLUSIONS: The results suggest that the neutrophilic infiltration in EIU occurs predominantly in the base of the ciliary body, whereas the monocytic and lymphocytic infiltrate occurs in the iris vasculature. Resident tissue macrophages do not undergo marked changes in density or morphology in the early course of the disease. Recruitment of T cells into the anterior segment in EIU may suggest a previously unsuspected role for these cells in the immunopathology of this disease. Changes in density and morphology of MHC class II+ DC in the iris, which persisted for at least 6 weeks, were interpreted as an increase in recruitment and migration of these cells that may serve to enhance the efficiency of immune surveillance in the anterior segment at crucial times of bacterial infection.