Immunohistochemical Double-staining Method Research Articles

N-myc downstream–regulated gene 1 can promote vasculogenic mimicry and angiogenesis in urothelial carcinoma

Urothelial carcinoma (UC) of the bladder is a common cause of cancer-related death worldwide. Vasculogenic mimicry (VM) is a process by which the malignant cells can generate vascular-like structures formed of periodic acid–Schiff (PAS) positive/CD31 negative extracellular matrix independent of angiogenesis and thus promotes tumor progression. N-myc downstream–regulated gene 1 (NDRG1) is a protein that can modulate tumor angiogenesis; however, its role in regulating tumor angiogenesis and VM formation has not been previously investigated in UC. This study aims to evaluate the role of intra-tumor microvessel density (MVD) (as a surrogate measure of angiogenesis), VM, and NDRG1 in UC and their correlation with different clinicopathologic features, then assess the correlation between them in UC. Sixty specimens of UC of the bladder were included. PAS-CD31 immunohistochemical double staining method was used to evaluate the intra-tumor MVD and VM. Immunohistochemical expression of NDRG1 was also examined. VM and NDRG1 expression were detected in 41.7% and 83.3% of UC specimens respectively. The mean of intra-tumor MVD, VM area, and NDRG1 was significantly higher in tumors with higher grade, lymphovascular invasion, and higher T stage. NDRG1 expression was positively correlated with MVD and VM. We can suggest that MVD, VM, and NDRG1 may serve as poor prognostic markers for UC. The positive correlation between NDRG1 and both MVD and VM may provide the first evidence that NDRG1 can induce tumor angiogenesis and VM in UC which may offer a novel pathway for further therapeutic strategies.

Visualisation of Microglia with the use of Immunohistochemical Double Staining Method for CD-68 and Iba-1 of Cerebral Tissue Samples in Cases of Brain Contusions.

In the recent years it has been confirmed that the main component of the immune response in an injury of the nerve cell comes from microglia and macrophages. The main challenge in the field of microglia research is to detect the different stages of cellular activation by visualization of the cell morphology. The existing visualization techniques are based on surface molecules expression in resting and activated microglia cells. For visualization of the microglial cells and their functional state we used double labeling method for cd-68 and iba1 in brain contusions with different survival time. Microglia are stained brown with Iba-1, whereas microglia impregnated with black, grainy color, represents activated microglia stained with CD 68. We had significantly positive results, and we were able to observe changes in the morphology of the microglia that correlated with the survival time. Using double labeling with Iba-1 and cd68 we were able to determine their physiological state based on the morphology and immunoreactivity.

Differential effects of experimental ulcerative colitis on P2X7 receptor expression in enteric neurons.

The digestive tracts of ulcerative colitis and Crohn's disease patients present with pathophysiological processes and intestinal necrosis. This study examined the P2X7 receptor and changes in the distal colon in enteric neurons of rats with experimental ulcerative colitis. The analysis was performed in the distal colons of rats with ulcerative colitis induced by the administration of 2,4,6-trinitrobenzene sulfonic acid (colitis group). The survival time after colitis induction was 24 h. The treated animals were compared to sham rats injected with phosphate-buffered saline and to animals with no intervention (control group). Tissues were prepared for immunohistochemical double-staining methods to examine P2X7 receptor, choline acetyltransferase (ChAT), calbindin, calretinin, anti-HuC/D (pan-neuronal) and S100β (pan-glial). The colocalization of the P2X7 receptor-immunoreactive (IR) cells was observed in the myenteric plexus with nitric oxide synthase (NOS)-, ChAT-,calbindin-, calretinin- and HuC/D-IR neurons and S100β-IR cells in the control, sham and colitis groups. The neuronal density (cell bodies/cm(2)) decreased in the myenteric plexus by 11, 18, 34, 22 and 60% in the P2X7 receptor, NOS-, ChAT-, calbindin- and calretinin-IR neurons, respectively. In addition, the densities (cell bodies/cm(2)) of HuC/D-IR neurons and S100β-IR enteric glial cells decreased by 33 and 29%, respectively. The profile areas were reduced by 6.8 and 21% in NOS- and ChAT-IR neurons, respectively. There was also a 20% increase of calbindin-IR neurons. Morphological changes were observed, such as increased neutrophils, disintegration of the intestinal epithelium and goblet cells and decreased collagen. This study demonstrated that colitis differentially affects P2X7 receptor-expressing enteric neurons based on their chemical codes and may cause changes in morphology and motility.

CD68+HLA-DR+ M1-like macrophages promote motility of HCC cells via NF-κB/FAK pathway

TAM is a prominent component of inflammatory microenvironment, presenting M1 and M2 polarized states in HCC. The objective of this study is to investigate the relationship between M1-polarized macrophages and metastasis in HCC. We used immunohistochemical double-staining method to inspect the infiltration of CD68+HLA-DR+ M1-like macrophages in HCC tissues. The M1-polarized macrophage was derived from THP-1 cell treated by LPS and IFN-γ in vitro. Transwell migration assay was used to evaluate whether the M1-polarized macrophage enhanced motility of HCC cells in the presence or absence of NF-κB inhibitor Bay 11-7802. The activation of NF-κB and FAK signaling pathways was examined by Western blot assay. Our results showed that the density of CD68+HLA-DR+ TAM in the HCC with metastasis is significantly higher than that in the HCC without metastasis. Moreover, the conditioned medium from the M1 macrophages promote the migration of HCC cells and induced the activation of NF-κB and FAK signaling. The promoted migration of HCC cells was abrogated by the Bay 11-7802, as well as the activation of NF-κB and FAK pathway. Our findings implied a pro-metastatic role of M1-like TAM in HCC.

Vitamin D-binding protein interacts with Aβ and suppresses Aβ-mediated pathology

The level of vitamin D-binding protein (DBP) is increased in the cerebrospinal fluid of patients with Alzheimer's disease (AD), suggesting a relationship with its pathogenesis. In this study, we investigated whether and how DBP is related to AD using several different approaches. A pull-down assay and a surface plasmon resonance binding assay indicated direct interactions between purified DBP and amyloid beta (Aβ), which was confirmed in the brain of AD patients and transgenic AD model mice by immunoprecipitation assay and immunohistochemical double-staining method. Moreover, atomic force microscopic examination revealed that DBP reduced Aβ aggregation in vitro. DBP also prevented Aβ-mediated death in cultured mouse hippocampal HT22 cell line. Finally, DBP decreased Aβ-induced synaptic loss in the hippocampus and rescued memory deficits in mice after injection of Aβ into the lateral ventricle. These results provide converging evidence that DBP attenuates the harmful effects of Aβ by a direct interaction, and suggest that DBP is a promising therapeutic agent for the treatment of AD.

Pituitary immunoexpression of ghrelin in anorexia nervosa

Ghrelin, an orexigenic hormone, is known to occur in the normal anterior pituitary where its physiologic role is uncertain but may include promotion of appetite. We sought to investigate anticipated differences in adenohypophysial and neurohypophysial ghrelin immunoexpression between normal subjects and patients with anorexia nervosa who had succumbed to complications of the disease. We hypothesized that the glands of anorexia nervosa patients would show relative diminished action in ghrelin content. The study included 12 autopsy-derived pituitaries of anorexia nervosa and 10 control glands. The streptavidin-biotin-peroxidase complex method and double immunohistochemical staining method were used to determine which cell types expressed both ghrelin and adenohypophysial hormones. Nontumorous control pituitaries were also obtained at autopsy. In anorexia nervosa and control adenohypophyses, ghrelin was mainly localized in somatotrophs and to a lesser extent in corticotrophs and gonadotrophs. Ghrelin accumulated within nerve fibers and Herring bodies in the neurohypophysis and pituitary stalk. In the controls, ghrelin expression was apparent in only a few cases. It was mild and only along few nerve fibers. In the adenohypophyses of anorexia nervosa patients, ghrelin was not depleted. It appears that in these patients, ghrelin is transported in excess from the hypothalamic neurohypophysial tract to the neurohypophysis.

Double Immunohistochemical Staining Method for HIF-1α and its Regulators PHD2 and PHD3 in Formalin-fixed Paraffin-embedded Tissues

Hypoxia-inducible factor (HIF-1alpha) is expressed in the nuclei of tumor cells under hypoxic conditions, and is regulated, in part, by cytoplasmic prolyl hydroxylases (PHDs). As HIF-1alpha is selectively expressed in tumor cells, inhibitors are being developed for cancer therapy. Although methods for the detection of HIF-1alpha and PHDs are available, an immunohistochemical double staining method for these markers in individual tumor cells is not available. For method development a human squamous cell carcinoma (SCC) xenograft, A253, was used as a known positive control tissue for HIF-1alpha in well-differentiated areas without microvessels. This laboratory showed that tumor cells in these areas are strongly positive for hypoxia markers. Another human, poorly differentiated SCC xenograft, FaDu, without hypoxic areas, was used as a negative control. PHD2 and 3 immunostaining was optimized individually using the human kidney. To optimize HIF-1alpha detection the pressure cooker time for antigen retrieval, concentration of the primary antibody, amplification reagent, and DAB development time were decreased. Casein blocking further decreased the background. Double staining resulted in brown nuclei for HIF-1alpha (DAB), and pink cytoplasmic staining for PHD2, 3 (fast red). The isotype-matched controls were negative. Normal human tissues had no detectable HIF-1alpha, but expressed PHD2, 3. The potential use of this new and improved method was confirmed by analyzing 15 surgical biopsies of oropharyngeal SCC of which 6 were positive for HIF-1alpha. This new method defined the optimal conditions for detection of HIF-1alpha and PHDs in individual tumor cells and could have a diagnostic and therapeutic potential.

Fas/Fas Ligand–mediated Apoptosis in Different Cell Lineages and Functional Compartments of Human Lymph Nodes

We have optimized an immunohistochemical double-staining method combining immunohistochemical lymphocyte lineage marker detection and apoptosis detection with terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling. The method was used to trace Fas-mediated apoptosis in human reactive lymph nodes according to cell lineage and anatomical location. In addition to Fas, we also studied the expression of Fas ligand (FasL), CD3, CD20, CD19, CD23, and CD68 of apoptotic cells. The presence of simultaneous Fas and FasL positivity indicated involvement of activation-induced death in the induction of paracortical apoptosis. FasL expression in the high endothelial venules might be an inductor of apoptosis of Fas-positive lymphoid cells. In addition to B-lymphocyte apoptosis in the germinal centers, there was often a high apoptosis rate of CD23-expressing follicular dendritic cells. In summary, our double-staining method provides valuable new information about the occurrence and mechanisms of apoptosis of different immune cell types in the lymph node compartments. Among other things, we present support for the importance of Fas/FasL-mediated apoptosis in lymph node homeostasis.

Immunohistochemical study of m1–m5 muscarinic receptors and nNOS in human inferior turbinate mucosa

Conclusion. This study suggested that nitric oxide (NO) takes part in the parasympathetic nerve control functions mainly through m3 receptors and subsequently through m1 receptors. Objectives. The regulation of glandular secretions and vasomotor tone in human nasal mucosa implicates muscarinic receptors. There are five recognized classes (m1–m5) of muscarinic receptor subtypes. NO is a free radical gas that has been found to be produced in neuronal cells by the action of enzyme neural nitric oxide synthase (nNOS). The aim of this study was to identify the colocalization of muscarinic receptor subtypes (m1–m5) and nNOS in the human inferior turbinate mucosa. Materials and methods. Human inferior turbinate mucosa was stained by using the antibody of muscarine receptor subtypes and nNOS in an immunohistochemical double-staining method. Results. The colocalization of m3 receptors and nNOS-immunoreactive nerve fibers was most extensively distributed on glands, vessels, and epithelium in all muscarinic receptor subtypes. These findings lead us to propose that NO also acts as a neurotransmitter in glands, vessels, and epithelium of the human nasal mucosa and can be assigned to parasympathetic nerve structures through m3 receptors mainly and m1 receptors subsequently.

Differential accumulation of pulmonary and cardiac mast cell-subsets and eosinophils between fatal anaphylaxis and asthma death: A postmortem comparative study

The distribution profile of infiltrated mast cell-subpopulations and eosinophils in the lung and heart sections of the patients who died of severe allergic hyperresponsiveness, was investigated. Four study groups were designed comprising 9 cases who died in systemic anaphylaxis (Group I), 10 asthmatic individuals whose death were assigned to acute and severe bronchial asthma (Group II), 10 asthmatic cases who died from non-immunological diseases (Group III). Twenty consecutive autopsies of non-allergic subjects who died of unnatural causes (Group IV) served as control group in this study. Utilizing antibodies against human tryptase and chymase and a double immunohistochemical staining method, we distinguished successfully all three subsets of mast cells (MC), MC-TC (containing both tryptase and chymase), MC-T (containing only tryptase) and MC-C (containing only chymase) types, subdivided on the basis of the protease compositions of their secretory granules. In order to immunostaining eosinophils, we used antibody to major basic protein as a marker. We also measured postmortem blood tryptase, specific and total serum IgE. The intriguing finding of this study was the marked differences of cellular composition in the lung between fatal anaphylaxis and asthma death. Significant augmentation of MCs infiltrated in lung and heart sections of anaphylaxis patients and drastic infiltration of bronchial eosinophils in asthmatic death and consequent release of their related inflammatory mediators might explain the differential expression of the associated symptoms in these two groups. The anaphylactic deaths did show neither emphysema nor significant mucous bronchial secretions whereas all asthmatic deaths did. The degree of pulmonary congestion and edema was also more severe in anaphylaxis. This corresponded with the histological findings and the location and number of mast cell-subsets and eosinophils in the different compartments of the lungs. We have demonstrated that the third type of mast cell MC-C is only found in the lungs in anaphylactic deaths. The practical consequence of our study will be that it is now possible to confirm a suspicion of anaphylaxis death not only by measurements of serum mast cell tryptase, but also by immunohistochemical methods.

IgE-positive plasma cells are present in adenoids of atopic children

Conclusions. We demonstrated the presence of IgE+ plasma cells in the adenoids of atopic children. Our data suggest that adenoids are capable of local production of IgE and support the role of adenoids as an inductive site for allergic reactions. Objective. We have previously demonstrated increased numbers of IgE+ cells in the adenoids of atopic children and also found support for an IL-4-induced class switch to IgE production in adenoids. In search of further evidence of adenoids being involved in IgE-mediated sensitization, we investigated the distribution of plasma cells and macrophages positive for IgE in adenoids. Material and methods. Adenoid tissue from atopic and non-atopic children was examined using immunohistochemical markers for IgE, plasma cells (CD138) and macrophages (CD68). The distribution of positive cells was determined in the extrafollicular area and in the follicles of adenoids. Co-localization of IgE and CD138+ plasma cells and CD68+ macrophages was examined using immunohistochemical double-staining methods. Results. Non-atopic adenoids contained no or very few IgE+ cells. In contrast, IgE+ cells were found in high numbers in atopic adenoids, mainly in the extrafollicular area. Higher numbers of IgE+ plasma cells and IgE+ macrophages were also found in the adenoids of atopic children.

Hepatic stellate cells in hepatitis C patients: relationship with liver iron deposits and severity of liver disease.

To determine the relationship between hepatic stellate cell (HSC) populations and severity of liver disease and liver iron deposits in patients with chronic hepatitis C virus (HCV). We also studied the relationship between iron cellular distribution and HSC population and the role of HFE mutations in the determination of iron deposits. Forty-nine chronic HCV patients with varying degrees of liver damage and liver iron deposits were studied. A liver biopsy was scored for histology activity index (HAI), fibrosis and iron deposits. The number of HSC in the liver was evaluated by an immunohistochemical double-staining method to identify glial fibrillary acid protein (GFAP) and smooth muscle alpha-actin (alpha-SMA). The HSC population was significantly higher in HCV patients than in normal controls and was predominant in zones 1 and 3. Liver iron deposits were observed in 49% of patients and were mild/moderate in most cases. We found a significantly higher number of GFAP and alpha-SMA positive cells in patients with liver iron deposits compared with those without iron deposits, and a positive correlation between liver iron scores and number (%) of GFAP and alpha-SMA positive cells. We observed a significantly higher number of GFAP and alpha-SMA positive cells in moderate/severe hepatitis than in minimal/mild hepatitis, and a positive correlation between GFAP and alpha-SMA positive cells and HAI and fibrosis scores. Liver iron deposits in chronic HCV are common and are associated with activation of HSC. Thus, even mild iron deposits might stimulate HSC and contribute to liver damage.

Actions of insulin-like growth factor binding protein-5 (IGFBP-5) are potentially regulated by tissue kallikrein in rat brains

Tissue kallikrein (EC 3.4.21.35) that hydrolyzes kininogen and releases a physiologically active peptide, kinin, is found in rat brains. Insulin-like growth factor binding proteins (IGFBPs) that modulate IGF actions are also expressed in a variety of tissues including rat brains, and one of the major IGFBPs expressed in brain is known to be IGFBP-5, which is reported to be hydrolyzed in vitro by prostate-specific antigen (PSA) and γ-nerve growth factor (γ-NGF), both of which belong to the member of the kallikrein gene family. This study was designed to determine whether or not kallikrein has a potential to hydrolyze IGFBP-5 and their topographic proximity was investigated in rat brain using double immunohistochemical staining method. Immunohistochemically, IGFBP-5 positive cells were numerous and widespread in the cerebral cortex and belonged to neurons in the cell configuration. IGFBP-5 positive cells were negative for S-100 protein and were positive for βIII tubulin, confirming them to be neurons. In addition, kallikrein positive cells were virtually all IGFBP-5 positive cells. IGFBP-5 was clearly hydrolyzed by kallikrein with cleavage sites of Arg 188-Met 189 and Arg 136-Arg 137 of IGFBP-5. Therefore, there is a possibility that kallikrein plays an important role in brain physiology, specifically in the neurons by regulating the actions of IGFBP-5 and IGF.

Vanilloid receptor homologue, VRL1, is expressed by both A- and C-fiber sensory neurons.

The capsaicin receptor (VR1) homologue, VRL1, is thought to be responsible for transducing high-threshold heat responses in Adelta-fiber neurons. In the present study, the expression of VRL1 by A- or C-fiber sensory neurons in rats was investigated by using a VRL1 and 200 kDa neurofilament (NF200, an A-fiber marker) double immunohistochemical staining method. Approximately 46% of VRL-positive neurons were NF200 positive. Though double-labeled neurons tended to be medium to large, many VRL1 single-labeled neurons were large. Dense VRL1 immunoreactivity was also found in laminae I and II of the spinal dorsal horn, where nociceptive Adelta- and C-fibers normally terminate. These results suggest that both C-fiber and Adelta-fiber primary sensory neurons express VRL1, and VRL1 may play an important role in nociception.

Three-dimensional morphology of c-Kit-positive cellular network and nitrergic innervation in the human gut.

-c-Kit-positive interstitial cells of Cajal (ICC) appear to play a key role in the normal motility function and development of intestine. Nitric oxide is considered to be the most important messenger of inhibitory nonadrenergic, noncholinergic nerves in the enteric nervous system. The aims of this study were to examine the distribution of nitrergic innervation and ICCs in normal human bowel and to demonstrate interconnections between ICCs and nitrergic nerves and smooth muscle fibers using histochemical and immunohistochemical double-staining methods with a whole-mount preparation technique and confocal laser scanning microscopy. Full-thickness small and large bowel specimens were obtained at autopsy from 18 children who died of nongastrointestinal diseases. A whole-mount preparation was performed for all specimens, and double staining was carried out with nicotinamide adenine dinucleotide phosphate (reduced form, NADPH)-diaphorase and c-Kit immunohistochemistry. Double immunofluorohistochemistry with neuronal nitric oxide synthase and c-Kit using confocal laser scanning microscopy was also performed in all specimens. The whole-mount preparation facilitated 3-dimensional visualization of the meshlike network of NADPH-diaphorase-positive nerve fibers in the myenteric plexus surrounded by a reticular network of c-Kit-positive ICCs. The dense c-Kit-positive cellular network located between longitudinal and circular muscle layers and at the innermost part of circular muscle layer intermingled with the myenteric plexus. Short, fine processes of ICCs made connections with the muscle fibers and c-Kit-positive cells. The development of double-NADPH-diaphorase histochemistry and c-Kit immunohistochemistry staining technique in a whole-mount preparation provides an easy and useful method for investigating the association between c-Kit-positive cellular network and nitrergic neuronal network in the human bowel wall. The characteristic profiles of the c-Kit-positive cellular network and nitrergic neuronal network and their relationship with the smooth muscle fibers provide a morphologic basis for investigating intestinal motility disorders.

Apoptosis and expression of p53 response proteins and cyclin D1 after cortical impact in rat brain

We measured the temporal profile and cellular identification of apoptosis in rat brain after cortical contusion injury. Double staining immunohistochemistry was also used to investigate the relationship between apoptotic cell death and selective protein expression associated with DNA damage and repair (p53, Bax, MDM2, WAF1, Gadd45, PCNA) and cell cycle protein, Cyclin D1, in male Wistar rats 48 h after injury. Cortical contusion was induced in male Wistar rats with a pneumatic impactor device. The animals were sacrificed at different times after trauma (1, 2, and 14 h and 1, 2, 4, 7 and 14 days; n=4 per time point). Sham-operated rats ( n=4) and normal rats not subjected to any surgical procedure ( n=4) were used as controls for temporal profile determination. Additional 11 rats were used for study of protein expression. Coronal brain sections were analyzed using an in situ terminal deoxynucleotdyl transferase-mediated biotinylated deoxyuridine triphosphate nick end labeling (TUNEL), hematoxylin, and immunohistochemical double staining methods. Apoptotic cells were observed as early as 2 h after the impact. Apoptotic cell death peaked at 2 days, gradually tapering off afterward, although scattered apoptotic cells were detected at 2 weeks after the impact. The number of apoptotic cells at 2 days far exceeded their number at other times ( p=0.009). Apoptotic cells were observed primarily in the cortex adjacent to the site of injury. In addition, apoptotic cells in conjunction with few injured cells were present in the ipsilateral hippocampus and localized to the granule layer of dentate gyrus. Our data indicate that DNA fragmentation is present in nearly all neurons subacutely after cortical contusion and persists for at least 2 weeks thereafter. Apoptosis is also present in neurons localized to the hilus of the dentate gyrus at a site remote from the area of injury suggesting a selective role for apoptosis in promoting secondary brain damage and dysfunction after traumatic brain injury. Using double staining, we were able to show that a great majority of apoptotic cells (>95%) were neurons and the rest were astrocytes and endothelial cells. Proteins associated with DNA damage and repair (p53, Bax, MDM2, WAF1, Gadd 45, PCNA) were expressed in the cytoplasm of normal cells of naive and sham rats. These proteins were translocated to the nuclei of apoptotic and injured cells at 48 h after cortical contusion. Cyclin D1 was not present in apoptotic cells. The differential expression of proteins associated with DNA damage, repair and the cell cycle protein Cyclin D1 in the contused brain suggest a potential role for these proteins in cell survival and apoptosis after cortical contusion.

Langerhans cells in prurigo nodularis investigated by HLA-DR and S-100 immunofluorescence double staining

The Langerhans cell is one of the antigen-presenting cells in the immune system. To study the presence of cutaneous Langerhans cells in prurigo nodularis, age- and sex-matched prurigo nodularis patient and healthy volunteer skin biopsies were investigated by an HLA-DR and S-100 immunohistochemical double staining method. The results showed that the HLA-DR- and S-100-immunoreactive (IR) Langerhans cells were altered in prurigo nodularis epidermis and dermis. The number of epidermal Langerhans cells in the prurigo nodularis patients was decreased in five and increased in two cases. In the dermis, the HLA-DR- and S-100-IR cells were apparently more numerous than in the controls. In the involved skin there were also more S-100-IR coarse nerve fibres in the dermis as compared to controls. The results indicate that dermal Langerhans cells (HLA-DR and S-100 double-labeled) as well as other dermal HLA-DR- and S-100-IR dendritic cells, but most likely not epidermal Langerhans cells, may be critically involved in the development or persistence of prurigo nodularis.

Cell dynamics of dividing cells in the olfactory epithelium of guinea pigs, using the immunohistochemical double staining method

It is thought that olfactory receptor cells regenerate after degeneration caused by various factors and that these cells undergo turnover under normal circumstances in the animal olfactory epithelium. Furthermore, the precursors of olfactory receptor cells were previously thought to be basal cells. Recently, however, it was reported that cells located just above the basal cells are the precursor cells on the basis of observation of dividing cells in the normal olfactory epithelium using methods such as bromodeoxyuridine (BrdU) uptake. In this study, the author observed the cell dynamics of dividing cells in the olfactory epithelium of guinea pigs by labeling the dividing cells with BrdU. Many of the BrdU-labeled cells migrated to the superficial layers of the olfactory epithelium, but a few of these cells remained just above the basal cells and did not express cytokeratin. Furthermore, the BrdU-labeled cells definitely matured to olfactory receptor cells. BrdU-labeled cells expressed a neural cell adhesion molecule (N-CAM) from 1 to 35 days after being labeled with BrdU. These cells also expressed protein gene product 9.5 (PGP 9.5), which is a kind of neuron-specific protein, from 5 to 35 days after being labeled with BrdU. From these results, it was concluded that the precursors of olfactory receptors cells were not basal cells. In other words, the lineage of olfactory receptor cells did not involve basal cells. Immature olfactory receptor cells expressed N-CAM, and PGP 9.5 as well as N-CAM were expressed with maturation.

Heterogeneity of mouse thymic macrophages: I. Immunohistochemical analysis.

As the first step toward understanding the identity and functions of thymic macrophages in situ, we examined the phenotypic heterogeneity of mouse thymic macrophages in tissue sections by the immunohistochemical double staining method with four monoclonal antibodies (F4/80, Mac-2, anti-CD32/16 and anti-I-A antibodies) as macrophage markers. Morphologically, three types of macrophages were identified: dendritic, round and flat-shaped. Dendritic macrophages were scattered throughout the thymus, and most of them were stained by all four markers. Among these macrophages, those at the cortico-medullary region (CMR) expressed a high intensity of CD32/16 antigen. Round macrophages were also distributed throughout the thymus; most of them, however, were localized in the cortico-medullary region to the medulla. These cells were F4/80-negative, Mac-2-positive, CD32/16-negative and I-A-positive. In contrast, round macrophages located at the cortex expressed F4/80. Flat-shaped macrophages were localized at the subcapsular region of the cortex where active lymphopoiesis was observed. This type was positive for F4/80 and CD32/16, but negative for Mac-2. Furthermore, most of the three types of thymic macrophages showed intense reactions of the I-A antigen within the cytoplasm in addition to the expression of I-A antigen on the cell membrane. These results indicate that morphological characteristics of thymic macrophages at different locations reflect phenotypic variations detected in immunohistochemistry, and suggest that these different type macrophages may play distinct roles at various locations in thymocyte development in the thymus.

Induction of DNA Fragmentation After 10 to 120 Minutes of Focal Cerebral Ischemia in Rats

The induction of neuronal necrosis has been studied after various durations of transient middle cerebral artery (MCA) occlusion in the rat. The objective of the present study was to measure the numbers and anatomic distribution of cells exhibiting apoptotic bodies as an indication of DNA fragmentation and apoptotic cell death as a function of duration of transient MCA occlusion in the rat. The MCA of male Wistar rats (n = 24) was occluded for 10, 20, 30, 60, 90, and 120 minutes (n = 4 per group) with the use of an intraluminal monofilament, and reperfusion was instituted for 48 hours. DNA fragmentation was measured in paraffin sections with the use of a terminal deoxynucleotidyl-transferase (TdT)-mediated dUTP-biotin nick end-labeling (TUNEL) method. Adjacent sections were stained with hematoxylin and eosin for analysis of ischemic cell damage, and immunohistochemical double staining methods were used for cell identification. Sham-operated rats (n = 4) and normal rats not subjected to any surgical procedure (n = 4) were used as controls for apoptosis detection. Within 5-microns-thick coronal sections, DNA fragmentation was present in 0 to 3 apoptotic cells in each hemisphere of normal, sham-operated rats as well as in the contralateral hemisphere of ischemic rats. After 10 to 20 minutes of MCA occlusion, apoptotic cells exhibiting DNA fragmentation (10 to 20) increased in the regions of selective neuronal necrosis in the preoptic area and in the striatum. After 30 to 60 minutes of ischemia, scattered apoptotic cells (30 to 60) exhibited DNA fragmentation and expanded into areas of selective neuronal necrosis in the cortex. After 90 to 120 minutes of occlusion, groups of apoptotic cells (70 to 200, > 95% neurons) were primarily localized to the inner boundary zone of the infarct. A range of mild to severe ischemia-reperfusion stimuli induce internucleosomal DNA cleavage. The presence and anatomic location of apoptotic cells exhibiting DNA fragmentation after transient cerebral occlusion indicate that apoptosis accompanies neuronal necrosis.