Calretinin Staining Research Articles

Calretinin immunohistochemistry for the diagnosis of Hirschprung disease in rectal biopsies

In this study we aimed to evaluate the usability of calretinin staining in the diagnosis and exclusion of HD in 36 rectal biopsies. Through immunohistochemical examination, in of a total of 21 pediatric patients in whom ganglion cells were detected in first rectal biopsies and in re-biopsies, ganglion cells were seen through nuclear and cytoplasmic staining. In the lamina propria and superficial submucosa, staining of nerve fibers was detected in a granular pattern in varying intensities. Out of a total of 5 biopsies (including one re-biopsy) of non-HD patients, where ganglion cells could not be seen, the nerve fibers were all stained. On the other hand, in 10 HD patients, diagnosed by a colon pull through operation, calretinin staining was not detected in any area of the rectal biopsies except for the mast cells. We conclude that calretinin immunostaining for the diagnosis of HD is an easy and reliable method for use in daily practice.

Dynamic expression of calretinin in embryonic and early fetal human cortex.

Calretinin (CR) is one of the earliest neurochemical markers in human corticogenesis. In embryos from Carnegie stages (CS) 17 to 23, calbindin (CB) and CR stain opposite poles of the incipient cortex suggesting early regionalization: CB marks the neuroepithelium of the medial boundary of the cortex with the choroid plexus (cortical hem). By contrast, CR is confined to the subventricular zone (SVZ) of the lateral and caudal ganglionic eminences at the pallial-subpallial boundary (PSB, or antihem), from where CR+/Tbr1- neurons migrate toward piriform cortex and amygdala as a component of the lateral cortical stream. At CS 19, columns of CR+ cells arise in the rostral cortex, and contribute at CS 20 to the “monolayer” of horizontal Tbr1+/CR+ and GAD+ cells in the preplate. At CS 21, the “pioneer cortical plate” appears as a radial aggregation of CR+/Tbr1+ neurons, which cover the entire future neocortex and extend the first corticofugal axons. CR expression in early human corticogenesis is thus not restricted to interneurons, but is also present in the first excitatory projection neurons of the cortex. At CS 21/22, the cortical plate is established following a lateral to medial gradient, when Tbr1+/CR- neurons settle within the pioneer cortical plate, and thus separate superficial and deep pioneer neurons. CR+ pioneer neurons disappear shortly after the formation of the cortical plate. Reelin+ Cajal-Retzius cells begin to express CR around CS21 (7/8 PCW). At CS 21–23, the CR+ SVZ at the PSB is the source of CR+ interneurons migrating into the cortical SVZ. In turn, CB+ interneurons migrate from the subpallium into the intermediate zone following the fibers of the internal capsule. Early CR+ and CB+ interneurons thus have different origins and migratory routes. CR+ cell populations in the embryonic telencephalon take part in a complex sequence of events not analyzed so far in other mammalian species, which may represent a distinctive trait of the initial steps of human corticogenesis.

Calretinin and calbindin distribution patterns specify subpopulations of type I and type II spiral ganglion neurons in postnatal murine cochlea

As the first neural element in the auditory pathway, neurons in the spiral ganglion shape the initial coding of sound stimuli for subsequent processing. Within the ganglion, type I and type II neurons form divergent and convergent innervation patterns, respectively, with their hair cell sensory receptors, indicating that very different information is gathered and conveyed. Layered onto these basic innervation patterns are structural and electrophysiological features that provide additional levels of processing multifaceted sound stimuli. To understand the nature of this additional complexity of signal coding, we characterized the distribution of calretinin and calbindin, two regulators of intracellular calcium that serve as markers for neuronal subpopulations. We showed in acute preparations and in vitro that calretinin and calbindin staining levels were heterogeneous. Immunocytochemical analysis of colocalization further showed that high levels of staining for the two molecules rarely overlapped. Although varied amounts of calbindin and calretinin were found within each tonotopic location and neuronal type, some distinct subdistributions were noted. For example, calretinin levels were highest in neurons innervating the midcochlea region, whereas calbindin levels were similar across the entire ganglion. Furthermore, we noted that apical type II neurons, identified by antiperipherin labeling, had significantly lower levels of calretinin and higher levels of calbindin. We also established that the endogenous firing feature of onset tau of the subthreshold response showed a pattern related to quantified calretinin and calbindin staining levels. Taken together, our results suggest an additional dimension of complexity within the spiral ganglion beyond that currently categorized.

Calretinin as a diagnostic adjunct for ameloblastoma.

Background. Calretinin is a 29 kDa calcium-binding protein of the EF-hand family which is expressed in a variety of normal and tumorigenic tissues. Its expression in odontogenic epithelium during odontogenesis and in neoplastic odontogenic tissues has been demonstrated. Unicystic ameloblastoma poses a diagnostic challenge, as its histologic presentation can be sometimes mistaken for keratocystic odontogenic tumor (KCOT). This study was performed to assess the usefulness of calretinin as a confirmatory marker for ameloblastic tissue. Methodology. Total of 40 cases: 16 unicystic ameloblastoma, 4 multicystic ameloblastoma, and 20 KCOT, were evaluated immunohistochemically for the presence, localization, distribution, and intensity of calretinin expression. Statistical analysis was done using Chi-square test to intercompare the expression between ameloblastoma and KCOT. Results. Sixteen cases of ameloblastoma (12 unicystic, 4 multicystic) showed positive calretinin staining of ameloblastic epithelium and only one case of KCOT was positive for calretinin, with the positivity restricted to the stellate reticulum like epithelium. Intercomparison between two groups revealed statistically significant difference (P = 0.000). Conclusion. Calretinin appears to be a specific immunohistochemical marker for neoplastic ameloblastic epithelium and may be an important diagnostic adjunct in the differential diagnosis of ameloblastoma and KCOT.

Comment on ‘Existing prognostic models, but not neutrophil-to-lymphocyte ratio, are prognostic in malignant mesothelioma’

Sir, We feel compelled to comment on the article of Meniawy et al (2013) to provide perspective on the value of the neutrophil to lymphocyte ratio (NLR) as a prognostic indicator in patients with malignant pleural mesothelioma (MPM). The Western Australia-based authors of this article have concluded from their analysis that the NLR did not provide prognostic value, whereas the Cancer and Leukemia Group B (CALGB) and European Organisation for Research and Treatment of Cancer (EORTC) prognostic guides did. However, there are some flaws in the data that have not been adequately acknowledged and that might have had a major impact on the conclusions. The principal flaw was that although intended to be an analysis of 369 consecutive patients presenting to a single treatment centre, this number was reduced by 95 (26%) based on failure to meet fairly arbitrarily defined inclusion criteria of: availability of a full blood count within 90 days of diagnosis; cytologically or histologically confirmed diagnosis of MPM; absence of concurrent haematological malignancy and duration of follow-up >90 days. A majority of patients (64) were excluded on the basis of missing laboratory data (unspecified as to which). There was no attempt to compare the characteristics of those excluded with those included to determine comparability of populations. In addition, of the remaining 274 patients, 169 (46% of initial) were treated with chemotherapy, whereas 105 (28%) received no systemic chemotherapy. In spite of 28% of patients receiving no treatment at all, the median survival for the entire group was 13.3 months with a median of 15.3 months for the chemotherapy group. These data appear to show unusually good overall survivals and are suggestive of selection bias, possibly caused by the exclusion of the 95 patients. In our original study in consecutive patients receiving systemic chemotherapy for MPM (Kao et al, 2010), the median survival was very similar to that reported by Vogelzang et al (2003) in their phase III study that compared pemetrexed and cisplatin with cisplatin alone. The findings of Meniawy and colleagues are also contradictory to the findings of other investigators in regard to the prognostic significance of NLR in MPM and numerous investigators in other tumour types (Cedres et al, 2012, 2013; Pinato et al, 2012; Guthrie et al, 2013; Paramanathan et al, 2014); however, the contradictory nature of their own findings was not adequately highlighted or explanation attempted. We have recently presented the outcomes of prognostic factors in a large cohort of patients (n=913) based on the clinical and laboratory data extracted from the records of the Dust Diseases Board of New South Wales (NSW), where median survival of patients was 10 months (Linton et al, 2013). In this large population-based study including >90% of the NSW patients seeking compensation from 2002 to 2009, NLR>5 was again found to be an independent poor prognostic factor (HR=1.21; CI: 1.02–1.44; P=0.03) in multivariate analysis (624 patients in the model), along with non-epithelial histology, age >70 years, male gender, stage III/IV, platelet count ⩾400, haemoglobin >1 g dl−1 decrease, negative calretinin staining in tumour specimen, not receiving pemetrexed chemotherapy and not receiving extrapleural pneumonectomy (EPP). Although the clinical factors were not in the final multivariate model, performance status was indirectly assessed in the model by including patients who received chemotherapy and EPP. In addition, we felt that the interesting observation of the significant predictive value of normalisation of NLR after one cycle of chemotherapy was brushed over in the article. This confirmatory finding after our initial article (Kao et al, 2010), along with the recent study demonstrating normalisation of NLR (<5) predicting for a survival benefit of 7 months in a series of 118 patients participating in phase I trials (Pinato et al, 2014), suggests that prospective validation of NLR is warranted. Finally, there appears to be a misconception that we were seeking a universal prognostic marker that could guide treatment outcomes for all. The series investigated by us confirm that determination of the NLR is a relatively simple way to assess prognosis in certain groups of patients with MPM; however, (ongoing) prospective validation will teach us how to properly use this parameter in clinical practice.

Calretinin, β‐Tubulin Immunohistochemistry, and Submucosal Nerve Trunks Morphology in Hirschsprung Disease

The aim of this study was to investigate calretinin and β-tubulin immunohistochemical expression together with submucosal nerve trunks morphology in differently innervated segments of Hirschsprung disease (HD) and total colonic aganglionosis (TCA). A total of 25 cases (22 HD, 3 TCA) and 18 controls were processed for calretinin and β-tubulin immunohistochemistry. Sections representative of distal aganglionic, transition, and proximal ganglionic segments were evaluated by a visual grading score; β-tubulin was evaluated also by image analysis. Submucosal nerve trunks hypertrophy and hyperplasia were measured by citomorphology. The length of proximal segment was correlated to postoperative bowel function. Controls showed intense calretinin and β-tubulin staining. In HD and TCA, calretinin staining was related to the presence of ganglion cells: negative in distal, faint in transition, intense in proximal segment. β-Tubulin staining was weak in all of the segments of HD and negative in TCA. Hypertrophic and hyperplastic nerve trunks characterized aganglionic segment, and progressively decreasing nerve size was observed in transition and ganglionic segments. Transient postoperative constipation, soiling, or enterocolitis was present in 59% of patients with HD without clear relation to proximal segment length or presence of hypertrophic nerve trunks. Calretinin is a reliable marker of the presence of ganglion cells, and, together with nerve hypertrophy, it helps to identify the transition zone. Length and nerve size of proximal segment in resected specimen did not affect the postsurgical intestinal function. Reduced β-tubulin expression along the entire colonic tract, included proximal ganglionic segments, may represent a potential impairing factor for the enteric neural transmission.

Use of automated image analysis in evaluation of Mesothelioma Tissue Microarray (TMA) from National Mesothelioma Virtual Bank

The National Mesothelioma Virtual Bank (NMVB) was established to provide annotated biospecimens to the mesothelioma research community. The resource provides tissue microarrays (TMA) to evaluate the biomarkers along with a variety of other resected mesothelioma specimens. In this manuscript, we describe the immunohistochemical evaluation of the mesothelioma TMA with three key antibodies that are used in making the diagnosis of mesothelioma, and compared the immunohistochemical assessment between manual scoring and image analysis.The TMA was assessed for the immunohistochemical expression of calretinin (N=39), cytokeratin (CK) 5/6 (N=33), and D2-40 (N=37). Immunohistochemistry was evaluated by semi-quantitative (manual) scoring using light microscope (MS) and by automated image analysis (AS).Calretinin staining was seen in both cytoplasmic and nuclear locations. CK5/6 stain was localized to the cytoplasm. D2-40 stain showed only membranous expression in our cases.•Based on the pathologist's scores, calretinin was positive in 31 of the 39 cases (80%), CK 5/6 in 15 of the 33 cases (46%) and D2-40 in 18 of the 37 cases (49%).•The percent-positive agreement between manual scores and image analysis was 90% (35/39), 94% (31/33), and 95% (35/37) for calretinin, CK 5/6, and D2-40, respectively. There was a substantial agreement between manual and automated scores for calretinin (kappa=0.614) and an almost perfect agreement for CK5/6 (kappa=0.879) and D2-40 (kappa=0.892).Our study confirms that the immunohistochemical staining pattern of mesotheliomas in the NMVB UPMC TMA is similar to other studies. Our findings also show that automated image analysis provides similar results to manual scoring by pathologists, and provides a reproducible, objective, and accurate platform for immunohistochemical assessment of biomarker expression.

Alteration of CaBP Expression Pattern in the Nucleus Magnocellularis following Unilateral Cochlear Ablation in Adult Zebra Finches

Songbirds have the rare ability of auditory-vocal learning and maintenance. Up to now, the organization and function of the nucleus magnocellularis (NM), the first relay of the avian ascending auditory pathway is largely based on studies in non-vocal learning species, such as chickens and owls. To investigate whether NM exhibits different histochemical properties associated with auditory processing in songbirds, we examined the expression patterns of three calcium-binding proteins (CaBPs), including calretinin (CR), parvalbumin (PV) and calbindin-D28k (CB), and their relations to auditory inputs in NM in adult zebra finches. We found enriched and co-localized immunostaining of CR, PV and CB in the majority of NM neurons, without neuronal population preference. Furthermore, they were sensitive to adult deafferentation with differential plasticity patterns. After unilateral cochlear removal, CR staining in the ipsilateral NM decreased appreciably at 3 days after surgery, and continued to decline thereafter. PV staining showed down-regulation first at 3 days, but subsequently recovered slightly. CB staining did not significantly decrease until 7 days after surgery. Our findings suggest that the three CaBPs might play distinct roles in association with auditory processing in zebra finches. These results are in contrast to the findings in the NM of chickens where CR is the predominant CaBP and deafferentation had no apparent effect on its expression. Further extended studies in other avian species are required to establish whether the difference in CaBP patterns in NM is functionally related to the different auditory-vocal behaviors.

Calretinin and microtubule-associated protein-2 (MAP-2) immunohistochemistry in the diagnosis of Hirschsprung's disease

Background/PurposeIdentifying ganglion cells by rectal suction biopsy is a basic diagnostic tool for the diagnosis of Hirschsprung's disease (HD). However, the difficult interpretation of conventionally processed slides often necessitates ancillary staining methods. The aim of this study was to evaluate the usefulness of calretinin and microtubule-associated protein-2 (MAP-2) immunohistochemistry in the diagnosis of HD. MethodsWe analyzed 52 rectal suction biopsy specimens (37 from 15 HD patients and 15 from 7 non-HD patients) for ganglion cells with calretinin and MAP-2 immunohistochemistry. We also analyzed full-thickness, frozen biopsy samples obtained from 15 HD patients who underwent surgery utilizing calretinin and MAP-2 immunohistochemistry. ResultsBoth calretinin and MAP-2 positively stained ganglion cells in the submucosal plexus of the ganglionic bowel but not aganglionic bowel. Calretinin usually stained ganglion cell cytoplasm and nuclei more intensely than MAP-2, which only stained cytoplasm. No nerve fiber staining in the submucosal layer was observed for either antibody. In 21.1% (11/52) of samples, calretinin and MAP-2 staining found ganglion cells which were reported not to have ganglion cells in the original surgical pathology reports. Immunohistochemical staining for calretinin using paraffin-embedded tissue sections after cryostat sections clearly demonstrated decreased staining intensity compared to MAP-2. ConclusionCalretinin and MAP-2 are useful diagnostic markers for diagnosing HD in rectal suction biopsies. These complementary methods could ameliorate the diagnostic difficulties associated with HD.

Hirschsprung Disease and Use of Calretinin in Inadequate Rectal Suction Biopsies

Up to 17% of all rectal suction biopsies performed in the evaluation of Hirschsprung disease are considered inadequate. In most instances, inadequate biopsies contain too little submucosa or are taken within the anal transition zone. To examine the utility of calretinin stain in the workup of inadequate biopsies for patients with clinical suspicion of Hirschsprung disease. A retrospective analysis was conducted of all rectal suction biopsies performed in the evaluation of Hirschsprung disease during the previous 12 years that were considered "inadequate." Seventeen cases were identified, and Hirschsprung disease status was determined by clinical or surgical follow-up. Immunohistochemistry for calretinin was performed for all cases containing columnar mucosa, which were evaluated without knowledge of clinical course. All 12 patients without Hirschsprung disease had calretinin-positive nerve fibers in the lamina propria or muscularis mucosae, and all 5 patients with Hirschsprung disease had no calretinin staining of nerves. In this retrospective series, calretinin immunohistochemistry correctly predicted outcome in all instances. Although the gold standard for the diagnosis of Hirschsprung disease in rectal suction biopsies remains the evaluation of ganglion cells in a hematoxylin-eosin staining with sufficient submucosa, calretinin immunohistochemistry is quite helpful in triaging further workup based on clinical suspicion.

A study of calretinin in Hirschsprung pathology, particularly in total colonic aganglionosis

IntroductionCalretinin, a calcium-binding protein, has been reported to be an important new marker in Hirschsprung's disease (HD). The aim is to study the diagnostic value of Calretinin in total colonic aganglionosis (TA), prematurity, and superficial biopsy when nerve hyperplasia may not be accessed by ACE activity. MethodsRecords of patients diagnosed with HD at our institution from 1985 to 2010 were studied and patients with TA identified. We examined tissue samples from those TA, partial colectomies for HD, biopsies for suspicion of HD, and rectal tissue from aborted fetuses. Immunohistochemical analysis of Calretinin was compared with ACE gold standard method in all cases. ResultsIn the majority of the cases, the diagnosis was ascertained by ACE activity and Calretinin staining. However, in 9 cases, the diagnosis was possible with Calretinin staining but not with ACE: in 4 TA because of the absence of nerve hyperplasia, and in 5 cases because the biopsies were too superficial to examine the nerve hyperplasia. In addition, Calretinin was expressed in the gut as early as 22 gestational weeks. ConclusionThe use of Calretinin staining may be superior to ACE activity, particularly in the context of TA, superficial biopsies, and prematurity, allowing earlier diagnosis.

Solitary Nasal Schwannoma: Usefulness of CD34 and Calretinin Staining for Distinction from Histological Mimics

A solitary nasal schwannoma in a 31-year-old woman is described. The patient had a 10-year history of left nasal obstruction and presented with worsening symptoms. Computed tomography revealed a mass in the left nasal cavity extending into the ethmoidal and sphenoidal sinuses. A large, white polypoidal mass with a smooth surface was removed endoscopically piece by piece. Microscopic examination showed the tumor to be a benign spindle-cell neoplasm with predominantly mixed cellular pattern and with an indistinct focal mixture of hypercellular and hypocellular areas, likely representing Antoni A and B areas, respectively. Differential diagnoses included schwannoma, neurofibroma, solitary fibrous tumor, and meningioma. On immunohistochemical examination, the neoplastic cells showed diffuse and strong positivity for S-100. CD34 was positive primarily in the hypocellular area (Antoni B) but weak or negative in the hypercellular area (Antoni A). Staining for calretinin was focal and strong, and that for glial fibrillary acidic protein was diffuse and weak; however, stainings for estrogen receptor, epithelial membrane antigen, and α-smooth muscle actin were negative. This immunohistochemical profile confirmed the diagnosis of schwannoma. The combined use of immunostains (CD34, calretinin) could be useful for differentiating sinonasal schwannoma from its histological mimics when the typical features are weak or absent.

26. Unusual adenomatoid tumour of the ovary consisting entirely of signet ring like-cells mimicking metastatic signet ring cell adenocarcinoma

Adenomatoid tumours of the ovary are rare benign neoplasms of mesothelial origin. They usually present as microscopic incidental findings in ovaries removed for other pathology. They are usually located within the ovarian hilum and tend to have a characteristic morphology of bland flattened to epithelioid cells with eosino-philic cytoplasm that are arranged into tubules, trabeculae and mi-crocystic spaces. We present an unusual case of an adenomatoid tumour arising wholly within the cortex of the ovary and consisting of entirely of infiltrative nests and small tubule-like structures composed of cells with a signet ring-like morphology. The im-munohistochemical profile of these cells was strong keratin posi-tivity and only weak, patchy staining for the mosethelial marker calretinin. The combination of these morphologic and immunohis-tochemical features could, under certain clinical conditions, particularly if a panel of appropriate immunohistochemical stains are not performed, lead to the over diagnosis of these benign tumours as metastatic signet ring cell adenocarcinoma. In view of the findings from our case, when considering a diagnosis of metastatic signet ring cell adenocarcinoma in the ovary, close attention must be paid to the cellular and nuclear features as well as the cytoplas-mic vacuoles to ensure that the diagnosis of adenomatoid tumour is not missed. We would also recommend using WT-1 and D2–40, in addition to calretinin, as additional evidence of mesothelial origin in a panel of immunohistochemical stains.

Significance of horizontal propagation of synchronized activities in human epileptic neocortex investigated by optical imaging and immunohistological study

To characterize the physiological condition of human epileptic neocortex, we employed flavoprotein fluorescence imaging (FFI), an optical imaging method which detects intrinsic signals accompanying neural activation, and immunohistologically studied human cortical specimens. The experimented materials were cortical tissues surrounding various intracerebral lesions obtained from 5 patients with epilepsy (epileptic patients: EPs) and 5 without epilepsy (non-epileptic patients: NEPs). These tissues were immersed in oxygenated artificial cerebrospinal fluid immediately after removal in the operating room. Signal changes of FFI in the cortical layers subjected to electrical stimulation were observed under bicuculline methiodide perfusion. Immunohistological staining for parvalbumin (PV), calbindin, and calretinin were performed on the same specimens to evaluate expressions of calcium-binding protein positive cells. The FFI study showed the characteristic cortical propagation pattern of elicited activities horizontally along the cortical layers in EPs but not in NEPs. The propagated area with more than 0.5% signal changes was significantly larger in EPs than in NEPs (p=0.008). Only the expression of PV positive neurons was significantly lower in EPs than in NEPs (p=0.006). The propagated area on FFI and the decrease in PV positive neurons correlated significantly (R=-0.78, p=0.04). The present study visualized the unique horizontal propagation of signal changes on FFI and demonstrated a correlation of this propagation with immunohistological decreases in PV positive neurons in human epileptic cortex. Further investigations may elucidate the mechanism of hyper-excitability and hyper-synchronization in epileptic cortical tissue itself.

A Case of Recurrent Sister Mary Josephʼs Nodule Related to Malignant Peritoneal Mesothelioma that Was Successfully Treated with Hyperthermic Intra-Peritoneal Chemotherapy (HIPEC)

Purpose: Malignant peritoneal mesothelioma is a highly lethal and aggressive tumor of the serosal membrane of peritoneum. Approximately 300-500 cases are diagnosed in USA every year and rarely do they involve the umbilicus. In most cases this is either due to metastatic disease or due to an umbilical hernia propagated by ascites and increased intrabdominal pressure in the course of diffuse malignant mesothelioma. We report a case of recurrent malignant peritoneal mesothelioma of epithelioid type presenting as a recurrent Sister Mary Joseph's nodule. A 50 year old Haitian male with a past medical history of gastric ulcer status post gastrojejunostomy who presented with recurrent periumbilical mass for 2-3 weeks with mild pain. The mass diameter size was 2 cm and slightly tender. 5 months prior to presentation, the patient had a similar mass at similar site which was surgically resected and sent for pathological examination which in addition to IHC staining (calretinin and CK5/6 Positivity) revealed malignant epithelioid mesothelioma. The patient subsequently received 4 cycles of permetrexed and cisplatin. PET scan revealed minimal change in size of the umbilical mass demonstrating hypermetabolic activity. There was an ametabolic region in the liver too. Patient was referred for direct inspection and underwent explorative laparotomy which showed invasive malignant peritoneal mesothelioma in right sided peritoneum, subdiaphragmatic peritoneum and right lateral peritoneum and patient underwent cytoreductive surgery, omentectomy, peritonectomy and hyperthermic intraperitoneal mitomycin c infusion. Patient is currently asymptomatic and doing well. Median survival for diffuse mesothelioma is 12-14 months but in recent trials, an intensive loco-regional treatment including cytoreductive surgery along with perioperative intra-peritoneal chemotherapy in the form of hyperthermic intra-peritoneal chemotherapy (HIPEC) with or without early post-operative intra-peritoneal chemotherapy (EPIC), were found to improve survival as evidenced in our interesting patient.Figure: Calretinin staining.

Morphine Decreases Enteric Neuron Excitability via Inhibition of Sodium Channels

Gastrointestinal peristalsis is significantly dependent on the enteric nervous system. Constipation due to reduced peristalsis is a major side-effect of morphine, which limits the chronic usefulness of this excellent pain reliever in man. The ionic basis for the inhibition of enteric neuron excitability by morphine is not well characterized as previous studies have mainly utilized microelectrode recordings from whole mount myenteric plexus preparations in guinea pigs. Here we have developed a Swiss-Webster mouse myenteric neuron culture and examined their electrophysiological properties by patch-clamp techniques and determined the mechanism for morphine-induced decrease in neuronal excitability. Isolated neurons in culture were confirmed by immunostaining with pan-neuronal marker, β-III tubulin and two populations were identified by calbindin and calretinin staining. Distinct neuronal populations were further identified based on the presence and absence of an afterhyperpolarization (AHP). Cells with AHP expressed greater density of sodium currents. Morphine (3 µM) significantly reduced the amplitude of the action potential, increased the threshold for spike generation but did not alter the resting membrane potential. The decrease in excitability resulted from inhibition of sodium currents. In the presence of morphine, the steady-state voltage dependence of Na channels was shifted to the left with almost 50% of channels unavailable for activation from hyperpolarized potentials. During prolonged exposure to morphine (two hours), action potentials recovered, indicative of the development of tolerance in single enteric neurons. These results demonstrate the feasibility of isolating mouse myenteric neurons and establish sodium channel inhibition as a mechanism for morphine-induced decrease in neuronal excitability.

FGF2 deficit during development leads to specific neuronal cell loss in the enteric nervous system

The largest part of the peripheral nervous system is the enteric nervous system (ENS). It consists of an intricate network of several enteric neuronal subclasses with distinct phenotypes and functions within the gut wall. The generation of these enteric phenotypes is dependent upon appropriate neurotrophic support during development. Glial cell line-derived neurotrophic factor (GDNF) and fibroblast growth factor-2 (FGF2) play an important role in the differentiation and function of the ENS. A lack of GDNF or its receptor (Ret) causes intestinal aganglionosis in mice, while fibroblast growth factor receptor signaling antagonist is identified as regulating proteins in the GDNF/Ret signaling in the developing ENS. Primary myenteric plexus cultures and wholemount preparations of wild type (WT) and FGF2-knockout mice were used to analyze distinct enteric subpopulations. Fractal dimension (D) as a measure of self-similarity is an excellent tool to analyze complex geometric shape and was applied to classify the subclasses of enteric neurons concerning their individual morphology. As a consequence of a detailed analysis of subpopulation variations, wholemount preparations were stained for the calcium binding proteins calbindin and calretinin. The fractal analysis showed a reliable consistence of subgroups with different fractal dimensions (D) in each culture investigated. Seven different neuronal subtypes could be differentiated according to a rising D. Within the same D, the neurite length revealed significant differences between wild type and FGF2-knockout cultures, while the subclass distribution was also altered. Depending on the morphological characteristics, the reduced subgroup was supposed to be a secretomotor neuronal type, which could be confirmed by calbindin and calretinin staining of the wholemount preparations. These revealed a reduction up to 40% of calbindin-positive neurons in the FGF2-knockout mouse. We therefore consider FGF2 playing a more important role in the fine-tuning of the ENS during development as previously assumed.

Transient Appearance of Tyrosine Hydroxylase Immunoreactive Cells in the Midline Epithelial Seam of the Human Fetal Secondary Palate

Transient immunoreactivity for tyrosine hydroxylase, which mediates the conversion of the amino acid L-tyrosine to dihydroxyphenylalanine, in the midline epithelial seam between the bilateral palatal shelves was investigated in human fetuses. Horizontal or frontal paraffin sections of two human fetuses at 9 and 15 weeks of gestation were used to examine the distribution of tyrosine hydroxylase-immunoreactive cells in regions of the entire head other than the brain. Immunohistochemical staining for S100 protein, calretinin, cytokeratin 14, and vimentin was examined using adjacent or near sections. Tyrosine hydroxylase-immunoreactive cells were large and densely distributed in the midline epithelial seam at the site of palatal fusion in fetuses at 9 weeks but not in fetuses at 15 weeks, in which the midline epithelial seam had already disappeared. No expression of S100 protein, calretinin, or vimentin was detected, but the midline epithelial seam was positive for cytokeratin 14. Tyrosine hydroxylase immunoreactivity was not detected in epithelia during the process of palatal fusion in mice from E 14.0 to 15.0. These findings indicate that tyrosine hydroxylase-immunoreactive cells in the midline epithelial seams are nonneural epithelial cells and suggest that the tyrosine hydroxylase is a novel factor involved in normal palatal formation, especially the fate of the midline epithelial seam in humans.

Calretinin immunohistochemistry in Hirschsprung’s disease: An adjunct to formalin-based diagnosis

This study was designed to assess the utility of calretinin immunohistochemistry in the diagnosis of Hirschsprung's disease. Eleven definitive resection materials from 10 Hirschsprung's disease patients and 3 initial full-thickness rectal biopsies of these patients were retrieved from the pathology archives. Additionally, 15 distal colon and 13 proximal colon full-thickness samples from 23 non-Hirschsprung's disease patients were also evaluated as the control group. All material was reevaluated by light microscopy for the presence or absence of ganglion cells and immunostained with calretinin, including proximal surgical margins and aganglionic zone samples from each resection material. Immunohistochemistry for calretinin provided highly compatible results with hematoxylin-eosin findings in Hirschsprung's disease and non-Hirschsprung's disease patients, except in one Hirschsprung's disease patient with very rare nerve stainings at the distal surgical margin. Calretinin immunohistochemistry was found to be highly sensitive and specific in detecting aganglionic segments. New research should be conducted in order to clarify calretinin staining patterns of the transitional zone, rare Hirschsprung's disease types, pure hypoganglionosis patients, and the anorectal junction, and for the mapping of fetal and neonatal colonic specimens. The technique seemed very effective for lowering the need for excessive sectioning and practical regarding the erratic nature of the acetylcholinesterase staining.

Extrapleural Pneumonectomy for Primary Pleural Mullerian Tumor in a Young Woman

We present a case of a low-grade mullerian tumor of right pleural origin in a 23-year-old woman treated with an aggressive multimodal treatment paradigm. This tumor is heretofore undescribed in the medical literature. She received induction therapy of carboplatin and paclitaxel with minimal response, then had a maximally cytoreductive right extrapleural pneumonectomy. She subsequently underwent intensity-modulated radiotherapy to the right hemithorax. We propose a treatment paradigm for this previously undescribed primary pleural mullerian tumor.