Thick Cryostat Sections Research Articles

Phrenic Nerve Transfer to Musculocutaneous Nerve: An Anatomical and Histological Study.

To restore elbow flexor muscle function in case of traumatic brachial plexus avulsion, the phrenic nerve transfer to the musculocutaneous nerve has become part of clinical practice. The nerve transfer can be done by means of video-assisted thoracic surgery without nerve graft or via supraclavicular approach in combination with an autograft. This study focuses on a detailed microscopic and macroscopic examination of the phrenic nerve. It will allow a better interpretation of existing clinical results and, thus, serve as a basis for future clinical studies. An anatomical study was conducted on 28 body donors of Caucasian origin (female n = 14, male n = 14). A sliding caliper and measuring tape were used to measure the diameter and length of the nerves. Sudan black staining was performed on 15 µm thick cryostat sections mounted on glass slides and the number of axons was determined by the ImageJ counting tool. In 23 individuals, the phrenic nerve could be examined on both sides. In 5 individuals, however, only one side was examined. Thus, a total of 51 nerves were examined. The mean length of the left phrenic nerves (33 cm (29-38 cm)) was significantly longer compared to the mean length of the right phrenic nerves (30 cm (24-33 cm)) (p < 0.001). Accessory phrenic nerves were present in 9 of 51 (18%) phrenic nerves. The mean number of phrenic nerves axons at the level of the first intercostal space in body donors with a right accessory phrenic nerve was significantly greater compared to the mean number of phrenic nerves axons at the same level in body donors without a right accessory phrenic nerve (3145 (range, 2688-3877) vs. 2278 (range, 1558-3276)), p = 0.034. A negative correlation was registered between age and the nerve number of axons in left (0.742, p < 0.001) and right (-0.273, p = 0.197) phrenic nerves. The mean distance from the upper edge of the ventral ramus of the fourth cervical spinal nerve to the point of entrance of the musculocutaneous nerve between the two parts of the coracobrachialis muscle was 19 cm (range, 15-24 cm) for the right and 20 cm (range, 15-25 cm) for the left arm. If an accessory phrenic nerve is available, it presumably should be spared. Thus, in that case, a supraclavicular approach in combination with a nerve graft would probably be of advantage.

Changes in Somatostatin-Like Immunoreactivity in the Sympathetic Neurons Projecting to the Prepyloric Area of the Porcine Stomach Induced by Selected Pathological Conditions.

The aim of the present study was to define changes in the expression of somatostatin (SOM) in the sympathetic perikarya innervating the porcine stomach prepyloric area during acetylsalicylic-acid-induced gastritis (ASA) and experimentally induced hyperacidity (HCL) and following partial stomach resection (RES). On day 1, the stomachs were injected with neuronal retrograde tracer Fast Blue (FB). Animals in the ASA group were given acetylsalicylic acid orally for 21 days. On the 22nd day after FB injection, partial stomach resection was performed in RES animals. On day 23, HCL animals were intragastrically given 5 ml/kg of body weight of a 0.25 M aqueous solution of hydrochloric acid. On day 28, all pigs were euthanized. Then, 14-μm thick cryostat sections of the coeliac-superior mesenteric ganglion (CSMG) complexes were processed for routine double-labelling immunofluorescence. All pathological conditions studied resulted in upregulation of SOM-like (SOM-LI) immunoreactivity (from 14.97 ± 1.57% in control group to 33.72 ± 4.39% in the ASA group, to 39.02 ± 3.65% in the RES group, and to 29.63 ± 0.85% in the HCL group). The present studies showed that altered expression of SOM occurs in sympathetic neurons supplying the prepyloric area of the porcine stomach during adaptation to various pathological insults.

Immunohistochemical colocalization of estrogen receptor-α and GABA in adult female rat hippocampus.

Hippocampus is an important target for estrogen action. It is severely affected in patients of Alzheimer's disease. Much of the current research related to estrogen and brain function is focused in two directions. By attempting to lacalize localizing ERs in GABAergic neurons of the hippocampus we tried to test the hypothesis that the action of estrogen in maintaining the neuronal plasticity and more specially the spine density of pyramidal neurons is through GABAergic neurons. The present study was planned to demonstrate the detailed immunoreactive (IR) distribution pattern of estrogen receptors (ER) in GABAergic neurons of hippocampus. The study was conducted in adult female Wistar rats in estrous phase. 30 µm thick cryostat sections of hippocampal region were obtained from perfusion fixed (with 4% buffered Para formaldehyde) adult female rats (n = 15). The sections were processed free- floating for immunolocalization using the PAP protocol. First they were immunostained for ER using, mouse monoclonal anti-ER-α antibody with DAB as chromogen. Subsequently the same sections were immunostained for GABA using rabbit monoclonal anti-GABA antibody respectively with 9 amino 3 ethyl carbazole (AEC) as chromogen. The results showed ER were colocalised in GABAergic neurons in all the subfields of hippocampus with obvious variations. In Cornua ammonis (CA) maximum co-localised neurons were seen in CA3 region. The view is strengthened by our results as it was established through previous studies that the immediate target neurons of estrogen in hippocampus is the GABAergic neurons.

Zinc transport through residual bodies in the rat testis; a histochemical study.

The aim of the present report was to study the zinc localization in rat testis with the help of a modified selective histochemical method (dithizone method). To achieve a selective demonstration of zinc, 16 microns thick serial cryostat sections of the Wistar rat testis were incubated with dithizone solution followed by a treatment with a "complexing buffer". PAS-reaction with haematoxylin was performed to determine the stages of spermatogenesis. During the maturation phase (from stage III onwards) the red coloured zinc dithizonate granules are observed in the mid piece and tail of elongated spermatids; the number and the stain intensity of zinc granules increase continuously till the stage VII. During the stages IX--X the zinc granules, present in the residual bodies, migrate from the adluminal to the basal Sertoli cell compartment. Thereafter, the zinc granules are observed only in the boundary tissue till the stage XIV. The results lead to suggest that the residual bodies play an essential role in the zinc transport.

Updated results on intestinal neuronal dysplasia (IND B): Meier-Ruge WA, Ammann K, Bruder E, et al. Eur J Pediatr Surg 2004 (December);14:384-391

Intestinal neuronal dysplasia (IND B) is still a subject of controversy. The aim of this paper was to review the present state of knowledge on IND B. A summary is given of the technical and diagnostic criteria which have to be considered to achieve a reliable diagnosis. The available therapeutic procedures are additionally discussed. Between 1992 and 2001, 3984 colonic mucosal biopsies from 1328 children were investigated. Nerve cell staining was performed on native tissue sections: 15 μm thick cryostat sections which, after spreading and drying, have a final thickness of 4-5 μm, with dehydrogenase reactions. The biopsies were taken 8-10 cm above the dentate line with a sufficient amount of submucosa. The criteria for IND B is 15-20% submucosa) giant ganglia with more than 8 nerve cells on 30 sections of a single biopsy. The diagnosis of IND B is quantitative. A diagnosis of IND B was made in 51 Hirschsprung resections, and in 92 children with chronic constipation (6% and 2.3% incidence, respectively). Up to the 14th year of life, most children with isolated IND B can be treated conservatively due to the delayed maturation of the enteric nervous system which is characteristic for IND B. Only children with additional hypo-plastic hypoganglionosis were treated surgically. Children with HD and IND B proximal to the aganglionosis often exhibited post-operative disturbances of intestinal motility when a disseminated IND B was present. Ganglioneuromatosis (MEN2B) must be clearly differentiated from IND B. The clinical course in IND B depends on the extent of impaired bowel innervation, the severity of motility affection and the coexistence of HD. Conservative management of isolated IND B is possible in most children. In individual cases, however, a transient enterostomy or segmental resection is unavoidable.

Updated Results on Intestinal Neuronal Dysplasia (IND B)

Intestinal neuronal dysplasia (IND B) is still a subject of controversy. The aim of this paper is to review the present state of knowledge on IND B. A summary is given of the technical and diagnostic criteria which have to be considered in order to arrive at a reliable diagnosis. In addition, the available therapeutic interventions are discussed. Between 1992 and 2001, 3984 colonic mucosal biopsies from 1328 children were investigated. Nerve cell staining was performed on native tissue sections: 15 microm thick cryostat sections, which, after spreading and drying on a microscopic slide, have a final thickness of 4-5 microm, with dehydrogenase reactions (lactic dehydrogenase, nitroxide synthase, succinic dehydrogenase). The biopsies were taken 8-10 cm above the dentate line (proximal to the ampulla recti, because of the caudo-cranial increase of giant ganglia proximal to the 4 cm biopsy) with a sufficient amount of submucosa. The criteria for IND is 15-20 % submucosal giant ganglia with more than eight nerve cells in 30 sections of a single biopsy (i.e. four to seven giant ganglia). The diagnosis of IND B is quantitative. A diagnosis of IND B was made over the past 10 years in 51 Hirschsprung resections (about 5 per year; 6 % of all Hirschsprung cases), and in 92 children with chronic constipation (about 9 children per year; 2.3 % incidence). Up to their fourth year of life, most children with isolated IND can be treated conservatively. This is due to the delayed maturation of the enteric nervous system which is characteristic of IND B. Only children who showed an additional hypoplastic hypoganglionosis were treated surgically. Children with Hirschsprung's disease (HD) and IND B proximal to the aganglionosis often showed, in those cases with a disseminated IND, postoperative disturbances in intestinal motility. The diagnosis of IND B requires that biopsies are taken proximal to the ampulla recti (about 8-10 cm above the dentate line) with a sufficient amount of submucosa. The biopsies must be cut rectangular to the surface of the mucosa. A diagnosis of IND B can be made only if, in the submucosa of 30 serial sections, 15-20 % of all ganglia are giant ganglia with more than eight nerve cells. Ganglioneuromatosis (MEN2B) must be clearly differentiated from IND. The clinical course of IND B depends on the extent of disturbed bowel innervation, the severity of motility failure, and the coexistence of MH. The conservative management of isolated IND is possible in most children. In individual cases, however, a transient enterostomy or a segmental resection is unavoidable.

Fibrogenesis and collagen resorption in the heart and skeletal muscle of Calomys callosus experimentally infected with Trypanosoma cruzi: immunohistochemical identification of extracellular matrix components.

Intense inflammatory lesions and early development of interstitial fibrosis of the myocardium and skeletal muscle with spontaneous regression, have been described in Calomys callosus infected with Trypanosoma cruzi. The genetic types of collagen present in this model were investigated through immunohistochemistry using specific antibodies, combined with histopathology and Picro-Sirius staining of collagen. Thirty-five calomys were infected with the Colombian strain of T. cruzi and sacrificed at 24, 30, 40, 60 and 90 days post-infection. Inflammatory lesions and fibrogenesis were prominent at the early phase of infection and significantly decreased during late infection. Immunoisotyping of the matrix components was performed by indirect immunofluorescence on 5 micro m thick cryostat sections using specific antibodies against laminin, fibronectin and isotypes I, III and IV of collagen. In the early phase, positive deposits of all the matrix components were present, with predominance of fibronectin, laminin and collagens types I and III in the myocardium and of types III and IV in the skeletal muscles. From the 40th day, type IV collagen predominates in the heart. At the late phase of infection (60th to 90th day), a clear fragmentation and decrease of all the matrix components were detected. Findings of the present study indicate that a modulation of the inflammatory process occurs in the model of C. callosus, leading to spontaneous regression of fibrosis independent of the genetic types of collagen involved in this process.

Quantitation of mRNA expression in glomeruli using laser-manipulated microdissection and laser pressure catapulting

Laser-manipulated microdissection (LMM) is a method to cut out a single cell or limited tiny region from a specimen under microscopic observation by a laser beam. Laser pressure catapulting (LPC) is a method to push up and collect samples that were microdissected using a strong laser. To induce experimental glomerulonephritis, anti-Thy1.1 monoclonal antibody (OX-7) was injected intravenously into rats. Control and disease model kidneys were obtained. Six-micrometer thick cryostat sections were mounted onto a 1.35 microm thin polyethylene membrane. Ten glomeruli were collected from 6 microm frozen sections of rat kidney by LMM and LPC. Isolated glomeruli were used to quantitate the expression of mRNA by real-time polymerase chain reaction (PCR). Transforming growth factor-beta1 (TGF-beta1) mRNA was not detected in glomeruli isolated by the LMM and the LPC methods on day 0, although G3PDH mRNA was measurable in the same samples. On day 7 after the treatment with OX-7, the ratio of TGF-beta1/G3PDH mRNA was 1.89 +/- 0.96 (N = 6). We established methods to isolate glomeruli from standard histochemical specimens by LMM and LPC, and to quantify mRNA expression in the targeted glomeruli using real-time PCR. We confirmed the up-regulation of TGF-beta1 mRNA expression in isolated glomeruli from frozen sections of the anti-Thy1.1 glomerulonephritis model.

Eosinophils are a feature of upper and lower airway pathology in non-atopic asthma, irrespective of the presence of rhinitis.

Asthma and rhinitis often co-exist and there are data to suggest that they may be two ends of the same disease spectrum. Immunohistochemical studies have shown that eosinophilia in the airways is a feature of rhinitic patients without asthma. The aim of our study was to examine whether cellular infiltration exists in the nasal mucosa of asthmatics even in the absence of symptoms and signs of rhinitis. Nasal mucosa biopsies were taken from 27 non-atopic subjects and comprised nine asthmatic rhinitic patients (AR), eight asthmatic non-rhinitic patients (ANR) and 10 healthy control subjects (N). Bronchial mucosa biopsies were also taken simultaneously from some of the patients (n = 10) to determine whether there was an association between cellular infiltration in the nose and the lungs. The alkaline phosphatase-anti-alkaline phosphatase (APAAP) method was used on 6 microm thick cryostat sections using monoclonal antibodies against T cells (CD4, CD8), eosinophils (EG2) and mast cells (mast cell tryptase). Slides were counted blind and results expressed as cells per field. The results showed that eosinophil counts were higher in both asthma groups compared with control nasal biopsies (median values AR 8.3, ANR 9.2, N 2.1 cells per field, P < 0.01). Furthermore, there was a significant correlation between eosinophil cell counts in the nose and the airways (r = 0.851 P < 0.001). No differences in eosinophil numbers were detected between the two groups of asthmatics. Also, no differences were noted for any other cell type (i.e. CD4, CD8, tryptase) among the three study groups. These results show that eosinophil infiltration was present in the nasal mucosa of asthmatic patients even in the absence of rhinitis, and add further support to the hypothesis that asthma and rhinitis are clinical expressions of the same disease entity.

La distribution des émetteurs α peu solubles déposés après inhalation dans les tissus pulmonaires : nouveaux abords méthodologiques

We have developed an autoradiograp hic method to provide representative images of dose rate and dose distribution in the lungs after actinide oxide inhalation. This was based on the study of 30 um thick dry sections obtained from 200 um thick cryostat sections. In this case, solid track detector had a recording yield similar to that obtain for 5 um thin sections and the resolution was less than 90 um. Using this method, we visualized a heterogeneous particle distribution as soon as 3 months after exposure of rats to (U, Pu)0 2 aerosols. Key-words - autoradiography, lungs, inhalation, actini.de oxides, dosimetry.

The expression of adhesion molecules in cigarette smoke-induced airways obstruction.

Cigarette smoking produces peripheral airway inflammation in all smokers, and chronic airways obstruction in approximately 20% of heavy smokers. The present study was designed to test the hypothesis that airways obstruction is related to changes in the expression of adhesion molecules involved in the recruitment of cells to sites of inflammation in the lung. Freshly resected lungs from heavy smokers with airways obstruction (n = 10) and from heavy smokers with normal lung function (n = 10) were collected in the operating room, inflated with optimal cutting temperature (OCT) medium and frozen over liquid nitrogen. Six micrometres thick cryostat sections cut from random samples of this tissue were stained, using immunohistochemistry, with monoclonal antibodies to the adhesion molecules on leucocytes: L-selectin, very late activation antigen-4 (VLA-4), CD11a/CD18, CD11b/CD18, CD11c/CD18; and on endothelial and epithelial surfaces: E-selectin, P-selectin, vascular cell adhesion molecule (VCAM-1), intercellular adhesion molecule (ICAM)-1 and ICAM-2 using the alkaline phosphatase anti-alkaline phosphatase (APAAP) technique. The slides were coded and the expression of each molecule scored by three observers using a semiquantitative grading system. Two inducible adhesion molecules, E-selectin on endothelium and CD11b on leucocytes, were also evaluated using quantitative morphometric analysis. The results showed a distribution of adhesion molecules that was consistent with the inflammatory response in the airways and parenchyma of all subjects but failed to show any differences between those with or without airways obstruction. We conclude that development of airways obstruction in heavy smokers cannot be explained by differences in the expression of adhesion molecules known to be involved in the control of cell traffic in the lung.

Vagal sensors in the rat duodenal mucosa: distribution and structure as revealed by in vivo DiI-tracing

Results from functional studies point to the importance of chemoreceptive endings in the duodenum innervated by vagal afferents in the regulation of gastrointestinal functions such as gastric emptying and acid secretion, as well as in the process of satiation. In order to visualize the vagal sensory innervation of this gut segment, vagal afferents were selectively labeled in vivo by injecting the lipophilic carbocyanine dye DiI into either the left or the right nodose ganglion of young adult rats. Thick cryostat sections or whole-mounted peels of muscularis externa or submucosa of formalin-fixed tissue were analyzed with conventional and/or confocal microscopy. In the mucosa, many DiI-labeled vagal afferent fibers were found with terminal arborizations mainly between the crypts and the villous lamina propria. In both areas, vagal terminal branches came in close contact with the basal lamina, but did not appear to penetrate it so as to make direct contact with epithelial cells. Labeled vagal afferent fibers in the villous and cryptic lamina propria were found to be in intimate anatomical contact with fibrocyte-like cells that may belong to the class of interstitial cells of Cajal, and with small granular cells that might be granulocytes or histiocytes. Although our analysis was not quantitative, and considering that labeling was unilateral and not complete, it appears that the overall density of vagal afferent mucosal innervation was variable; many villi showed no evidence for innervation while other areas had quite dense networks of arborizing terminal fibers in several neighboring villi. Analysis of separate whole-mounted muscularis externa and submucosa peels revealed the presence of large bundles of labeled afferent fibers running within the myenteric plexus along the mesenteric attachment primarily in an aboral direction, with individual fibers turning towards the antimesenteric pole, and either penetrating into the submucosa or forming the characteristic intraganglionic laminar endings (IGLEs). Although the possibility of individual fibers issuing collaterals to myenteric IGLEs and at the same time to mucosal terminals was not demonstrated, it cannot be ruled out. These anatomical findings are discussed in the context of absorptive mechanisms for the different macronutrients and the implication of enteroendocrine cells such as CCK-containing cells that may function as intestinal "taste cells".

Regeneration pattern of blood vessels and nerves in cultured keratinocyte grafts assessed by confocal laser scanning microscopy.

The aim of this study was to investigate the pattern of both neovascularization and reinnervation, and the relationship between the two processes, in keratodermal grafts, using confocal laser scanning microscopy, at different time points during the healing process. Keratodermal grafts were prepared in pigs by combining autologous dermis with cultured autologous keratinocytes. Immunohistochemistry was carried out on thick cryostat sections (100-150 microns), using antisera to the endothelial marker von Willebrand factor (vWf) and the pan-neuronal marker protein gene product 9.5 (PGP9.5). The results suggest that the neovascularization and reinnervation in the cultured keratodermal graft is almost complete at 6 weeks. Neovascularization precedes innervation, reaching the surface covered by the keratinocytes at 2 weeks, initially with a linear vascular pattern. From 3 weeks, there is a gradual arborization of the vessels to form a typical vascular plexus. The process of reinnervation is similar in pattern to that of neovascularization, although slower in developing a full network of fibres. In conclusion, the use of confocal microscopy allows the precise definition of complex patterns of neovascularization and nerve growth, which are not fully apparent when using conventional microscopy. Because angiogenesis occurs first, it probably plays a leading role in the survival of keratodermal grafts during wound healing. Indeed, new blood vessels form a pathway for the subsequent innervation process, and quickly reach the epidermal layer which, in turn, may play a key role in the tropism of both blood vessels and nerves.

Vagal innervation of the rat pylorus: an anterograde tracing study using carbocyanine dyes and laser scanning confocal microscopy.

In an attempt to identify the distribution and structure of vagal fibers and terminals in the gastroduodenal junction, vagal efferents were labeled in vivo by multiple injections of the fluorescent carbocyanine dye DiA into the dorsal motor nucleus (dmnX), and vagal afferents were anterogradely labeled by injections of DiI into the nodose ganglia of the same or separate rats. Thick frontal cryostat sections were analysed either with conventional or laser scanning confocal microscopy, using appropriate filter combinations and/or different wavelength laser excitation to distinguish the fluorescent tracers. Vagal efferent terminal-like structures were present in small ganglia within the circular sphincter muscle, which, in the absence of a well-developed, true myenteric plexus at this level, represent the myenteric ganglia. Furthermore, vagal efferent terminals were also present in submucosal ganglia, but were absent from mucosa, Brunner's glands and circular muscle fibers. Vagal afferent fibers and terminal-like structures were more abundant than efferents. The most prominent afferent terminals were profusely branching, large net-like aggregates of varicose fibers running within the connective tissue matrix predominantly parallel to the circular sphincter muscle bundles. Profusely arborizing, highly varicose endings were also present in large myenteric ganglia of the antrum and duodenum, in the modified intramuscular ganglia, and in submucosal ganglia. Additionally, afferent fibers and terminals were present throughout the mucosal lining of the gastroduodenal junction. The branching patterns of some vagal afferents suggested that individual axons produced multiple collaterals in different compartments. NADPH-diaphorase positive, possibly nitroxergic neurons were present in myenteric ganglia of the immediately adjacent antrum and duodenum, and fine varicose fibers entered the sphincter muscle from both sides, delineating the potential vagal inhibitory postganglionic innervation. These morphological results support the view of a rich and differentiated extrinsic neural control of this important gut region as suggested by functional studies.

Modification of standard proteinase K/phenol method for extraction of DNA from small tumour biopsies

The standard proteinase K/phenol DNA isolation method was found to produce unsatisfactory yields of DNA from small tissue biopsies (less than 50 mg). The influences of the volume of cell lysis buffer and the amount of proteinase K on the final DNA yield and quality were studied, and an improved method was devised and compared with both the standard procedure and a phenol-free protocol. The optimal volume of cell lysis buffer was found to be 200 microliters per mg of tissue while the optimal amount of proteinase K was 60 micrograms per mg of tissue. A mean yield of 12 mu/mg tissue of pure, high molecular weight DNA was achieved from 50 frozen samples prepared by crushing. Yields from 20 microns thick cryostat sections reached 30 micrograms/mg.

Alkaline phosphatase and peptidase levels in invertebrate cartilage.

Cartilage is encountered in the skeletons of many advanced invertebrates, yet it never calcifies or is replaced by bone. In an attempt to account for the absence of bone in invertebrates, we tested a hypothesis proposing that absence or inadequate quantities of several enzymes associated with vertebrate osteogenesis may underlie the failure of the invertebrates to evolve bone. The enzymes examined were alkaline phosphatase, alanyl beta-naphthylamidase, and neutral protease. Their activities were measured in the gill cartilage of the Atlantic horseshoe crab, Limulus polyphemus, and the odontophore cartilage of the marine whelk, Busycon canaliculatum. Animals were collected from the Cape Cod area. Samples of cartilage of Limulus perichondrium, various non-skeletal tissues, and neonatal rat calvaria, the latter as a reference standard, were homogenized in 0.1 M phosphate buffer (pH 7.1) and analyzed for protein content and the above-mentioned enzyme activities. Alkaline phosphatase specific activity was readily detected in most tissues except the invertebrate cartilage specimens in which it was present only at near-trace levels. Naphthylamidase and protease activities were present in all tissues. In a single experiment, higher phosphatase values were recorded for Limulus cartilage retaining perichondrium, but in a subsequent trial assaying cartilage retaining perichondrium, denuded cartilage, and isolated perichondrium separately, it was demonstrated that phosphatase activity resided primarily within the perichondrium. Exposure of thick cryostat sections to p-nitrophenyl phosphate confirmed the suspicion that alkaline phosphatase activity was present principally in the perichondrium.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunolocalization of ecto-5′-nucleotidase in the kidney by a monoclonal antibody

A monoclonal antibody IgG, has been raised against ecto-5'-nucleotidase purified from rat kidney homogenate. The specificity of the antibody was verified by immunoprecipitation. The distribution of the corresponding antigen in the rat kidney was studied by immunocytochemistry (FITC and PAP technique) in 1 micron thick cryostat sections. The antibody reacted with the brush border of proximal tubules, the apical cell membrane and the apical cytoplasm of intercalated cells in connecting tubules and collecting ducts and with interstitial cells of the cortex. Among the interstitial cells exclusively stellate shaped fibroblasts were reactive whereas rounded interstitial cells (type II interstitial cells) as well as pericytes and endothelial cells of peritubular capillaries were unreactive. Compared to the staining intensity of the fibroblasts in the cortical labyrinth the reactivity of the fibroblasts in the medullary rays of the cortex was weak or absent. Interstitial cells of the entire medulla were unreactive. Concerning the fibroblasts in the periarterial connective tissue, those surrounding the larger arteries (arcuate arteries, cortical radial arteries) were negative, those alongside afferent and efferent arterioles were positive. Endothelia of lymphatic capillaries travelling within the periarterial connective tissue were also positive. All components of the juxtaglomerular apparatus were negative. The findings are consistent with an interstitial production of adenosine, available extracellularly and thus being able to reach the major target sites of adenosine, the smooth muscles of glomerular arterioles, including the granular cells at the glomerular vascular pole.

Immunocytochemical characterization of supporting cells in the enteric nervous system in Hirschsprung's disease

The enteric nervous system (ENS) is composed of two distinct neural components, extrinsic and intrinsic, and its supporting cells uniquely possess some characteristics of both central nervous system (CNS) astrocytes and peripheral nervous system (PNS) Schwann cells. To provide further insight into the neural defects in Hirschsprung's disease, the supporting cells in biopsied normal gut, ganglionic, and aganglionic segments from six cases of Hirschsprung's disease were investigated immunocytochemically for localization of three neuroglial markers, glial fibrillary acidic protein (GFAP), S-100 protein, and glutamine synthetase (GS), by the avidin-biotin-horseradish peroxidase complex method applied to free-floating thick cryostat sections. In normal control gut and ganglionic segments of Hirschsprung's colon, all of the GFAP, S-100, and GS were expressed strongly by the supporting cells of the myenteric and submucosal plexuses, interconnecting nerve fiber bundles of the plexuses, and fine nerve strands in the muscular layer. The nerve bundles of the subserosa merging into the muscular layer were also immunoreactive for GFAP and S-100, but negative or only faintly positive for GS. On the other hand, aberrantly proliferated nerve bundles in the aganglionic segment of the Hirschsprung's colon were accompanied by supporting cells strongly positive for GFAP and S-100, but negative or faintly positive for GS. These results indicate that the supporting cells of the enteric neurons proper, enteric glia, express GFAP, S-100, and GS, whereas the supporting cells of the extrinsic components, which accompany PNS axons, are negative or very weakly positive for GS. Thus, GS immunocytochemistry may delineate intrinsic and extrinsic neural components in the ENS, and may provide an important clue for differential diagnosis of Hirschsprung's disease.

Three-dimensional visualization of human Langerhans' cells using confocal scanning laser microscopy

Most techniques used to visualize cells in tissues are accompanied by some distortion of the tissue due to fixation and sectioning. We here present a technique involving "optical sectioning" and three-dimensional reconstruction of fluorescence-labelled Langerhans' cells in human skin that avoids such problems. The instrument used to study the specimens was a PHOIBOS Confocal Scanning Laser Microscope (CSLM) built around a Zeiss Universal microscope. A software package for three-dimensional reconstructions of confocal sections was employed for calculations in a Hewlett-Packard HP9000-350 minicomputer running UNIX. Normal human skin obtained at plastic surgery or skin biopsy specimens from allergic, irritant, or control patch test reactions was studied. Epidermis separated from dermis by incubation in thermolysin or thick (25 microns) cryostat sections were incubated with fluorescein-isothiocyanate labelled mouse monoclonal antibodies directed against HLA-DR. To reduce the fading of immunofluorescence during microscopy, the specimens were mounted with glycerol in PBS containing p-phenylenediamine. The results indicate that CSLM is a powerful tool for investigations on Langerhans' cells in different conditions of the skin allowing a three-dimensional view of cells in unfixed or acetone-fixed preparations at the light microscope level.

Tissue specificity in cytochrome c oxidase deficient myopathy

Biopsied muscles were treated in 2 ways to demonstrate cytochrome c oxidase (CCO) activity on electron microscopy: (1) one to several muscle fibers were teased off the biopsy in buffer solution after glutaraldehyde fixation, (2) 20–30-μm thick cryostat sections were placed on precooled glass slides and fixed in glutaraldehyde solution at room temperature. After rinsing in buffer, the teased fibers and cryostat sections were stained with cytochrome c oxidase. In both procedures, almost all mitochondria in control muscle fibers stained positively. In CCO deficiency, the enzyme activity differed from tissue to tissue indicating marked tissue specificity. In the fatal infantile form enzyme activity in muscle fibers was absent, but present in fibroblasts, endothelial cells and smooth muscle arterial cells. The enzyme activity in other forms differed from cell to cell, but individual mitochondria in a given cell examined in cross-section showed uniform CCO activity, indicating that there was no intracellular mosaicism of enzyme positive and negative mitochondria.