Acetylcholinesterase Histochemical Staining Research Articles

Application of 3D Image Technology in Motor and Sensory Nerve Classification

Objective. To explore the application of 3D image technology in motor and sensory nerve classification. Methods. A total of 200 sections of the 5cm-long popliteal fossa peroneal nerve from adult volunteers were cut and frozen. The slices were 10 m thick, and the interval between the slices was 0.25 mm. Acetylcholinesterase histochemical staining was used to observe the changes of nerve bundles under the microscope. The stained sections were transformed into digital images by the digital camera system, and the images were stitched to obtain a two-dimensional panoramic image 100 times magnified. The properties of the functional bundles were manually judged. Using Amira 3.1 three‐dimensional reconstruction software to realize the three-dimensional reconstruction and visualization of nerve can not only accurately perceive the complex three-dimensional surface structure of nerve, but also arbitrarily display, rotate, scale, and segment the three-dimensional structure inside nerve, and carry out three-dimensional measurement in time. It has made preliminary achievements in brachial plexus, lumbosacral plexus, neural stem functional bundle (group), and intramuscular nerve routing and distribution, including the regeneration process of sensory nerve and three-dimensional reconstruction and visualization of composite tissue containing sensory nerve. Conclusion. Based on histology and computer technology, the functional band of short peroneal nerve can be reconstructed in 3D, which provides a feasible basis for the three-dimensional reconstruction of the functional band of the long peripheral nerve.

An α7-related nicotinic acetylcholine receptor mediates the ciliary arrest response in pharyngeal gill slits of Ciona.

Ciliary movement is a fundamental process to support animal life, and the movement pattern may be altered in response to external stimuli under the control of nervous systems. Juvenile and adult ascidians have ciliary arrays around their pharyngeal gill slits (stigmata), and continuous beating is interrupted for seconds by mechanical stimuli on other parts of the body. Although it has been suggested that neural transmission to evoke ciliary arrest is cholinergic, its molecular basis has not yet been elucidated in detail. Here, we attempted to clarify the molecular mechanisms underlying this neurociliary transmission in the model ascidian Ciona Acetylcholinesterase histochemical staining showed strong signals on the laterodistal ciliated cells of stigmata, hereafter referred to as trapezial cells. The direct administration of acetylcholine (ACh) and other agonists of nicotinic ACh receptors (nAChRs) onto ciliated cells reliably evoked ciliary arrest that persisted for seconds in a dose-dependent manner. While the Ciona genome encodes ten nAChRs, only one of these called nAChR-A7/8-1, a relative of vertebrate α7 nAChRs, was found to be expressed by trapezial cells. Exogenously expressed nAChR-A7/8-1 on Xenopus oocytes responded to ACh and other agonists with consistent pharmacological traits to those observed in vivo Further efforts to examine signaling downstream of this receptor revealed that an inhibitor of phospholipase C (PLC) hampered ACh-induced ciliary arrest. We propose that homomeric α7-related nAChR-A7/8-1 mediates neurociliary transmission in Ciona stigmata to elicit persistent ciliary arrest by recruiting intracellular Ca2+ signaling.

Distribution characteristics of sweat gland nerve fibres in normal humans identified by acetylcholinesterase histochemical staining

ObjectiveTo quantitatively analyze distribution characteristics of sweat gland nerve fibres (SGNF) in normal Chinese individuals for obtaining a reference for early diagnosis of peripheral neuropathy. Patients and methodsSkin biopsy samples were collected from 192 normal Chinese individuals and divided into six, four and two groups according to anatomic sites, age and gender, respectively. SGNF morphology was observed and SGNF density (SGNFD) was determined. ResultsThere was a significant difference in SGNFD among different anatomic sites, age and gender. A degressive tendency was observed from proximal to distal anatomic sites. SGNFD was the lowest in subjects in the 21–40-year-old age group, but was the highest in subjects in the >61-year-old age group. Overall, SGNFD fluctuated with age. SGNFD in males was significantly higher than that in females. ConclusionsDistribution characteristics of SGNF in normal individuals may serve as a reference for early diagnosis of nerve fibre damage.

Audit of diagnoses of neonatal rectal aganglionosis demonstrates little role for acetylcholinesterase histochemical staining

Audit of diagnoses of neonatal rectal aganglionosis demonstrates little role for acetylcholinesterase histochemical staining

Innervation of the rabbit cardiac ventricles.

The rabbit is widely used in experimental cardiac physiology, but the neuroanatomy of the rabbit heart remains insufficiently examined. This study aimed to ascertain the architecture of the intrinsic nerve plexus in the walls and septum of rabbit cardiac ventricles. In 51 rabbit hearts, a combined approach involving: (i) histochemical acetylcholinesterase staining of intrinsic neural structures in total cardiac ventricles; (ii) immunofluorescent labelling of intrinsic nerves, nerve fibres (NFs) and neuronal somata (NS); and (iii) transmission electron microscopy of intrinsic ventricular nerves and NFs was used. Mediastinal nerves access the ventral and lateral surfaces of both ventricles at a restricted site between the root of the ascending aorta and the pulmonary trunk. The dorsal surface of both ventricles is supplied by several epicardial nerves extending from the left dorsal ganglionated nerve subplexus on the dorsal left atrium. Ventral accessing nerves are thicker and more numerous than dorsal nerves. Intrinsic ventricular NS are rare on the conus arteriosus and the root of the pulmonary trunk. The number of ventricular NS ranged from 11 to 220 per heart. Four chemical phenotypes of NS within ventricular ganglia were identified, i.e. ganglionic cells positive for choline acetyltransferase (ChAT), neuronal nitric oxide synthase (nNOS), and biphenotypic, i.e. positive for both ChAT/nNOS and for ChAT/tyrosine hydroxylase. Clusters of small intensely fluorescent cells are distributed within or close to ganglia on the root of the pulmonary trunk, but not on the conus arteriosus. The largest and most numerous intrinsic nerves proceed within the epicardium. Scarce nerves were found near myocardial blood vessels, but the myocardium contained only a scarce meshwork of NFs. In the endocardium, large numbers of thin nerves and NFs proceed along the bundle of His and both its branches up to the apex of the ventricles. The endocardial meshwork of fine NFs was approximately eight times denser than the myocardial meshwork. Adrenergic NFs predominate considerably in all layers of the ventricular walls and septum, whereas NFs of other neurochemical phenotypes were in the minority and their amount differed between the epicardium, myocardium and endocardium. The densities of NFs positive for nNOS and ChAT were similar in the epicardium and endocardium, but NFs positive for nNOS in the myocardium were eight times more abundant than NFs positive for ChAT. Potentially sensory NFs positive for both calcitonin gene-related peptide and substance P were sparse in the myocardial layer, but numerous in epicardial nerves and particularly abundant within the endocardium. Electron microscopic observations demonstrate that intrinsic ventricular nerves have a distinctive morphology, which may be attributed to remodelling of the peripheral nerves after their access into the ventricular wall. In conclusion, the rabbit ventricles display complex structural organization of intrinsic ventricular nerves, NFs and ganglionic cells. The results provide a basic anatomical background for further functional analysis of the intrinsic nervous system in the cardiac ventricles.

A combined acetylcholinesterase and immunohistochemical method for precise anatomical analysis of intrinsic cardiac neural structures

A significant challenge when investigating autonomic neuroanatomy is being able to reliably obtain tissue that contains neuronal structures of interest. Currently, histochemical staining for acetylcholinesterase (AChE) remains the most feasible and reliable method to visualize intrinsic nerves and ganglia in whole organs. In order to precisely visualize and sample intrinsic cardiac nerves and ganglia for subsequent immunofluorescent labeling, we developed a modified histochemical AChE method using material from pig and sheep hearts. The method involves: (1) chemical prefixation of the whole heart, (2) short-term and weak histochemical staining for AChE in situ, (3) visual examination and extirpation of the stained neural structures from the whole heart, (4) freezing, embedding and cryostat sectioning of the tissue of interest, and (5) immunofluorescent labeling and microscopic analysis of neural structures. Firstly, our data demonstrate that this modified AChE protocol labeled intrinsic cardiac nerves as convincingly as our previously published data. Secondly, there was the added advantage that adrenergic, cholinergic and peptidergic neuropeptides, namely protein gene product 9.5 (PGP 9.5), neurofilament (NF), tyrosine hydroxylase (TH), vesicular monoamine transporter (VMAT2), neuronal nitric oxide synthase (nNOS), choline acetyltransferase (ChAT), calcitonin gene related peptide (CGRP), and substance P may be identified. Our method allows the precise sampling of neural structures including autonomic ganglia, intrinsic nerves and bundles of nerve fibers and even single neurons from the whole heart. This method saves time, effort and a substantial amount of antisera. Nonetheless, the proof of specific staining for many other autonomic neuronal markers has to be provided in subsequent studies.

A.P.13: Myotonic discharges in autophagic vacuolar myopathies: A potential electrophysiologic marker

Disordered lysosomal function within muscle cells is the basis for a group of diseases referred to as autophagic vacuolar myopathies. These disorders include acid maltase deficiency (Pompe disease), LAMP-2 deficiency (Danon disease), and X-linked myopathy with excess autophagy (XMEA). We present a 65year old diabetic woman with bilateral leg weakness evolving over 2years to the point she was unable to ambulate without assistance. There was no family history of a similar disorder. She had prominent weakness in her legs proximally>distally while arms and bulbar muscles had normal strength. Mild percussion myotonia of forearm muscles was present but not hand grasp myotonia. CK was 914. Acid alpha-glucosidase activity was normal. Nerve conduction studies showed evidence of a largely axonal neuropathy likely related to her poorly controlled diabetes. Needle exam demonstrated fibrillations and frequent short duration myotonic discharges in arm and leg muscles. Quadriceps muscle biopsy showed basophilic cytoplasmic stippling within myofibers that corresponded to small vacuoles discernable by enzyme histochemical staining for acetylcholinesterase and immunostaining for dystrophin and other sarcolemmal proteins. Cytoplasmic clusters of lysosomes and autophagosome extrusion with duplication of basement membranes were observed with electron microscopy. The biopsy findings are those of an autophagic vacuolar myopathy, the underlying nature of which has not been defined. Frequent short duration myotonic discharges were a remarkable feature. The presence of myotonic discharges has been previously reported to occur in patients with Pompe disease, Danon disease, and XMEA. These discharges indicate hyperexcitability of the muscle membranes and may reflect structural sarcoplasmic changes. Their occurrence in the various disorders involving autophagy suggests that short myotonic discharges may be a diagnostically useful electrophysiologic marker for this group of disorde.

Neuroanatomy and immunohistochemistry of cardiac ventricles in rabbit (543.2)

Rabbit model is used in cardiac electrophysiology but neuroanatomy of the rabbit heart (RH) is poorly examined. We aimed to ascertain the architecture of intrinsic cardiac nerve plexus of the RH, and the neurochemical phenotypes of ventricular nerves (VNs) and nerve fibers (NFs) using histochemical acetylcholinesterase staining and immunofluorescent labeling for PGP 9.5, TH, ChAT, nNOS, and SP in 18 New Zealand white rabbits. Cardiac nerves (CNs) accessed ventricles of the RH by the left and middle dorsal and the right and left coronary subplexuses. In the rabbit ventricular walls, there were found TH, ChAT, nNOS and SP positive (+) NFs. In VNs, more than 65% of NFs were TH(+), while only 7% were cholinergic ones. On the average, 4% of nNOS(+) and SP(+) NFs were found in VNs. The TH(+) NFs also predominated in the interventricular septum, papillary muscles, and in perivascular nerves. The PGP9.5(+) NFs distributed within the myocardium occupied 4.2% in the left ventricle and 3.8% in the right one. The mean area of NFs in papillary muscle were 2.6% for TH(+), 2.4% for ChAT(+), 1.4% for SP(+) and 0.6% for nNOS(+). Endocardial nerves contained 15.8% TH(+), 6.3% SP(+), 4.2% ChAT(+), and 1.5% nNOS(+) NFs. In summary, the RH corresponds neuroanatomically to hearts of some rodents, but it’s noticeably distinct from the human one.Grant Funding Source: MIP‐031/2013 from the Research Council of Lithuania

A new diagnostic scoring system to differentiate Hirschsprung’s disease from Hirschsprung’s disease-allied disorders in patients with suspected intestinal dysganglionosis

To create a simple diagnostic scoring system to differentiate Hirschsprung's disease (HD) from Hirschsprung's disease-allied disorders (HAD) in patients with suspected intestinal dysganglionosis (IDs). Between 1998 and 2008, 967 patients with suspected intestinal dysganglionosis underwent surgical treatment at the pediatric surgery department of Tongji Hospital. The diagnosis of HD or HAD was confirmed by postoperative pathological examination. All patients underwent preoperative work-up including barium enema, anorectal manometry, and histochemical acetylcholinesterase staining of rectal mucosa. Known risk factors for IDs were recorded. The predicting score was calculated by summing the scores of the risk factors and three preoperative tests. The sensitivity, specificity, accuracy, positive predictive values, negative predictive values, positive likelihood ratios, and negative likelihood ratios of the predicting score were calculated. The cutoff score for predicting HD was determined using receiver operating characteristic (ROC) analysis. The accuracy of the predicting score was measured by the area under the ROC curve. Failed or delayed passage of meconium, age <3 years and male gender were risk factors associated with HD. The area under the ROC curve of the predicting score was 0.927 (95 % confidence interval, 0.910-0.944). A predicting score of more than 5 was used as a cutoff for predicting HD. The scoring system achieved 83.1 % sensitivity, 89.5 % specificity, and 85.9 % accuracy in predicting HD. Patients with a predicting score of more than 5 are more likely to be diagnosed with HD, whereas a score less than 5 are mostly indicative of HAD.

Neuroanatomy of the murine cardiac conduction system

The mouse heart is a popular model to study the function and autonomic control of the specialized cardiac conduction system (CCS). However, the precise identity and anatomical distribution of the intrinsic cardiac nerves that modulate the function of the mouse CCS have not been adequately studied. We aimed at determining the organization and distribution of the intrinsic cardiac nerves that supply the CCS of the mouse. In whole mouse heart preparations, intrinsic neural structures were revealed by histochemical staining for acetylcholinesterase (AChE). Adrenergic, cholinergic and peptidergic neural components were identified, respectively, by immunohistochemical labeling for tyrosine hydroxylase (TH), choline acetyltransferase (ChAT), calcitonin gene related peptide (CGRP), substance P (SP), and protein gene product 9.5 (PGP 9.5). Myocytes of the CCS were identified by immunolabeling of hyperpolarization activated cyclic nucleotide-gated potassium channel 4 (HCN4). In addition, the presence of CCS myocytes in atypical locations was verified using fluorescent immunohistochemistry performed on routine paraffin sections. The results demonstrate that four microscopic epicardial nerves orientated toward the sinuatrial nodal (SAN) region derive from both the dorsal right atrial and right ventral nerve subplexuses. The atrioventricular nodal (AVN) region is typically supplied by a single intrinsic nerve derived from the left dorsal nerve subplexus at the posterior interatrial groove. SAN myocytes positive for HCN4 were widely distributed both on the medial, anterior, lateral and even posterior sides of the root of the right cranial (superior caval) vein. The distribution of HCN4-positive myocytes in the AVN region was also wider than previously considered. HCN4-positive cells and thin slivers of the AVN extended to the roots of the ascending aorta, posteriorly to the orifice of the coronary sinus, and even along both atrioventricular rings. Notwithstanding the fact that cholinergic nerve fibers and axons clearly predominate in the mouse CCS, adrenergic nerve fibers and axons are abundant therein as well. Altogether, these results provide new insight into the anatomical basis of the neural control of the mouse CCS.

Galantamine as a Preventive of Diisopropylphosphorofluoridate Toxicity Effects in Rat Brain

Diisopropylfluorophosphate exerts its toxic effect by irreversibly inhibiting acetylcholinesterase. This results in over-stimulation of central and peripheral cholinergic activity. The aim of the present study was to evaluate the possible preventive effects of acute treatment with reversible acetylcholinesterase inhibitor galantamine against the signs of cholinergic toxic syndrome provoked by diisopropylfluorophosphate, such as hypothermia, muscular fasciculations, oral dyskinesia and decreased locomotor performance in a rat model of intoxication. The effects of these two anticholinesterases on acetylcholinesterase activity and on the expression of mRNA of the immediate early response gene c-fos in the brain were assessed by histochemical acetylcholinesterase staining and by in situ hybridization, respectively. Diisopropylfluorophosphate induced rapidly progressing hypothermia, muscular fasciculations, oral dyskinesia and decreased locomotor performance. The increased cholinergic cortical and hippocampal activity due to irreversible acetylcholinerase inhibition were indicated by the increased c-fos mRNA autoradiographic signal and by the inhibition of acetylcholinesterase staining, respectively. Galantamine by itself provoked transient and relatively weak inhibition of the acetylcholinesterase staining, while it did not induce increased c-fos mRNA expression or significant behavioural signs of cholinergic toxicity. Galantamine significantly reduced the rate of the onset, but not the maximal hypothermia induced by diisopropylfluorophosphate. Importantly, all the above-mentioned behavioural and neurochemical effects of diisopropylfluorophosphate were significantly reduced by galantamine. These results indicate that the acute pre-treatment with galantamine may have prophylactic effects against the intoxication by diisopropylfluorophosphate.

Morphological pattern of mouse intrinsic cardiac nerve plexus

The study was aimed to determine the 3D organization and immunohistochemistry of intrinsic neural plexus in mouse heart. In a whole heart preparation, intrinsic neural structures were revealed by a histochemical staining for acetylcholinesterase, while the double labeling of tyrosine hydroxylase (TH), choline acetyltransferase (ChAT), substance P (SP) and calcitonin gene related peptide (CGRP) was applied to identify immunohistochemically the distribution of adrenergic, cholinergic and peptidergic neural components, respectively. Our findings demonstrate that extrinsic cardiac nerves access the ganglionated nerve plexus of the mouse heart hilum at the right and left cranial veins. Nerves and bundles of nerve fibers extend epicardially from this plexus to atria and ventricles by 5 neural pathways or subplexuses. Generally, the examined hearts from 56 mice contained 19±3 ganglia, in which 1.082±160 neurons reside. Majority of intrinsic neurons (83%) are ChAT(+), 3% ‐ TH(+); 14% – bifenotypic, i.e. they were immunoreactive both for ChAT and TH. The TH(+) epicardial nerve fibers are predominant on the dorsal and ventral sides of left atrium, whereas the most ChAT(+) axons proceed on the heart base toward the large intrinsic ganglia and within epicardium of the root of the right cranial vein. Peptidergic nerve fibers were abundant in the epicardium and within intrinsic ganglia adjacent to the heart hilum. In conclusion, although majority of nerves and neural bundles in the mouse heart are mixed, some of them express either adrenergic or cholinergic phenotype. This study was supported by the Research Council of Lithuania (No. MIP‐11184).

Study of Sensory and Motor Fascicles in Brachial Plexus and Establishment of a Digital Three-Dimensional Graphic Model

To investigate a 3-dimensional (3D) model of human brachial plexus including its topography of sensory and motor fascicles with the assistance of the computer technology, 2 brachial plexus were serially horizontally sliced. Each slice was stained by Karnovsky-Roots acetylcholinesterase histochemical method. The stained sections were scanned, and the image was put into the computer serially. At last, the 3D diagram of brachial plexus was made. The internal structure of the brachial plexus was found to be very complicated. The fascicles bifurcated and recombined with one another with no fixed rules. All fascicles were mixed sensory and motor fibers. Acetylcholinesterase histochemical staining from a serial tissue section is a useful technique to distinguish sensory fibers from motor ones. The 3D visualization of the brachial plexus may help to develop a computerized database of the fascicle topography to provide an anatomical reference in fascicular repair of brachial plexus.

Application of Acetylcholinesterase Histochemistry for the Diagnosis of Hirschsprung’s Disease in Neonates and Infants: a Twenty-year Experience

The acetylcholinesterase (AChE) histochemical staining of intestinal mucosal-submucosal biopsy specimens is believed to be the most reliable diagnostic method for Hirschsprung's disease (HD). The aim of our study was to evaluate advantages and disadvantages of this method for HD diagnosis in infants and neonates. The results of AChE histochemistry of rectal biopsy specimens, obtained from 11 neonates and 29 infants treated in the Clinic of Pediatric Surgery, Hospital of the Lithuanian University of Health Sciences, from 1991 to 2010 were analyzed. AChE activity of neural structures was evaluated using Karnovsky-Roots method modified by El-Badawi and Schenk. Two neonates were diagnosed with HD. The diagnosis was not confirmed in 9 cases, but clinical symptoms progressed in 3 cases, and HD was diagnosed after the repeated biopsy performed in infancy. The results of primary biopsy were rated as false negative. Test sensitivity and specificity in neonates were 40.0% and 100%, respectively. A total of 21 infants were diagnosed with HD. All of them underwent surgery. The diagnosis of HD was confirmed in 20 cases; in one case, intestinal neuronal dysplasia type B was diagnosed. The diagnosis was not confirmed in 8 cases. In infants, the test had a sensitivity of 100% and a specificity of 88.8%. The analysis of AChE activity in rectal biopsy specimens is a reliable method for diagnosing HD in infancy. This test is less valuable in neonates. If test results are negative, infants should be observed, and if symptoms persist, the biopsy should be repeated at the age of 3 months. Rectal biopsy specimens in neonates should include mucosa and submucosa.

Calretinin immunostaining as an adjunct in the diagnosis of Hirschsprung disease

Historically, the diagnosis of Hirschsprung disease was made by evaluating multiple hematoxylin and eosin-stained slides and performing acetylcholinesterase histochemical staining. Recently, calretinin immunohistochemical staining has been reported and found to be superior to acetylcholinesterase staining in the confirmation of aganglionosis. We retrieved tissue blocks from 23 patients with proven Hirschsprung disease from the archives of the Medical College of Georgia. In addition, we selected 23 control patients with ganglion cells. All cases were stained with calretinin, and the presence or absence of both intrinsic nerve fibers (INFs) and ganglion cells was scored by 4 pathologists with fairly strong agreement (κ = 0.858). All cases of proven Hirschsprung disease were negative for INFs. Eighty-three percent of non-Hirschsprung patients were positive for INFs. Based on statistical analysis, the association between disease status and pathologist rating was statistically significant (P < .0001). We also found calretinin immunostaining to be a useful adjunctive modality in the diagnosis of Hirschsprung disease.

Morphologic pattern of the intrinsic ganglionated nerve plexus in mouse heart

Both normal and genetically modified mice are excellent models for investigating molecular mechanisms of arrhythmogenic cardiac diseases that may be associated with an imbalance between sympathetic and parasympathetic nervous input to the heart. The purpose of this study was to (1) determine the structural organization of the mouse cardiac neural plexus, (2) identify extrinsic neural sources and their relationship with the cardiac plexus, and (3) reveal any anatomic differences in the cardiac plexus between mouse and other species. Cardiac nerve structures were visualized using histochemical staining for acetylcholinesterase (AChE) on whole heart and thorax-dissected preparations derived from 25 mice. To confirm the reliability of staining parasympathetic and sympathetic neural components in the mouse heart, we applied a histochemical method for AChE and immunohistochemistry for tyrosine hydroxylase (TH) and/or choline acetyltransferase (ChAT) on whole mounts preparations from six mice. Double immunohistochemical labeling of TH and ChAT on AChE-positive neural elements in mouse whole mounts demonstrated equal staining of nerves and ganglia for AChE that were positive for both TH and ChAT. The extrinsic cardiac nerves access the mouse heart at the right and left cranial veins and interblend within the ganglionated nerve plexus of the heart hilum that is persistently localized on the heart base. Nerves and bundles of nerve fibers extend epicardially from this plexus to atria and ventricles by left dorsal, dorsal right atrial, right ventral, and ventral left atrial routes or subplexuses. The right cranial vein receives extrinsic nerves that mainly originate from the right cervicothoracic ganglion and a branch of the right vagus nerve, whereas the left cranial vein is supplied by extrinsic nerves from the left cervicothoracic ganglion and the left vagus nerve. The majority of intrinsic cardiac ganglia are localized on the heart base at the roots of the pulmonary veins. These ganglia are interlinked by interganglionic nerves into the above mentioned nerve plexus of the heart hilum. In general, the examined hearts contained 19 ± 3 ganglia, giving a cumulative ganglion area of 0.4 ± 0.1 mm(2). Despite substantial anatomic differences in ganglion number and distribution, the structural organization of the intrinsic ganglionated plexus in the mouse heart corresponds in general to that of other mammalian species, including human.

Utilization of peripherin and S-100 immunohistochemistry in the diagnosis of Hirschsprung disease

Evaluation of rectal biopsies for ganglion cells is performed for patients suspected of having Hirschsprung disease. At times, identification of ganglion cells can be difficult, especially in newborns. To assist in diagnosis, frozen tissue can be collected for acetylcholinesterase histochemical staining. At our institution, we developed a protocol using peripherin and S-100 immunostaining as an adjunct to hematoxylin and eosin (H&E) for the identification of ganglion cells. Further, at the time of frozen section, we performed Diff Quik staining to highlight ganglion cells. One hundred and thirty eight rectal biopsies submitted for evaluation of Hirschsprung disease were compiled from the archives of the Medical College of Georgia from 2002 to 2009. Initial evaluation consisted of eight levels of H&E-stained slides and two unstained slides each for immunostaining with peripherin and S-100. If on initial evaluation, ganglion cells were not identified, additional H&E and peripherin immunostains were performed. Peripherin immunostaining was unequivocally identified in the cytoplasm of ganglion cells of patients at all ages. Of the 136 patients with diagnostic biopsies, 80% had ganglion cells. Of these, 93% of cases were diagnosed on the original eight levels. Twenty-seven cases were devoid of ganglion cells, and of these, 81% showed submucosal neural hypertrophy on S-100 staining. Twenty-six patients had confirmed aganglionic segments at the time of colonic resection. One patient had colostomy only. A total of 54 frozen sections were performed on 25 patients over this same period of time. Diff Quick staining was found to be very useful. In this study, our protocol proved to be very sensitive, specific, and efficient for the diagnosis of Hirschsprung disease.

Donepezil Inhibits Diisopropylfluorophosphate-Induced Seizures and Up-regulation of Synaptotagmin 4 mRNA

Reversible acetylcholinesterase inhibitor donepezil displays prophylactic effects against intoxication with irreversible organophosphorous acetylcholinesterase inhibitors. We used behavioural observation of yawning and epileptic seizures, histochemical acetylcholinesterase staining, and in situ hybridization of the immediate early genes, c-fos and synaptotagmin 4 (Syt4) mRNAs in the brain, to evaluate whether donepezil could protect the brain against the effects of the organophosphate anticholinesterase, diisopropylfluorophosphate, in a rat model of intoxication. Diisopropylfluorophosphatetreated animals exhibited frequent yawning, significant inhibition of acetylcholinesterase staining and upregulation of c-fos mRNA, but not the epileptic seizures or significant change of Syt4 mRNA levels. In order to reduce the threshold for the induction of cholinergic seizures, additional groups of rats were pre-treated with LiCl 24 h before the treatment with diisopropylfluorophosphate. These rats exhibited the seizures, a significant inhibition of acetylcholinesterase staining and significant upregulation of c-fos and Syt4 mRNA levels. All the above-mentioned effects of diisopropylfluorophosphate were inhibited by donepezil pre-treatment. Donepezil pre-treatment by itself induced only a comparatively weaker inhibition of acetylcholinesterase staining and infrequent yawning. We conclude that donepezil protects the brain against diisopropylfluorophosphate-induced effects and that Syt4 mRNA upregulation may serve as a novel marker for organophosphate-induced seizures.

Amyloid-β expression in retrosplenial cortex of triple transgenic mice: relationship to cholinergic axonal afferents from medial septum

Triple transgenic (3×Tg–AD) mice harboring the presenilin 1, amyloid precursor protein, and tau transgenes (Oddo et al., 2003b) display prominent levels of amyloid-beta (Aβ) immunoreactivity in forebrain regions. The Aβ immunoreactivity is first seen intracellularly in neurons and later as extracellular plaque deposits. The present study examined Aβ immunoreactivity that occurs in layer III of the granular division of retrosplenial cortex (RSg). This pattern of Aβ immunoreactivity in layer III of RSg develops relatively late, and is seen in animals older than 14 months. The appearance of the Aβ immunoreactivity is similar to an axonal terminal field and thus may offer a unique opportunity to study the relationship between afferent projections and the formation of Aβ deposits. Axonal tract tracing techniques demonstrated that the pattern of axon terminal labeling in layer III of RSg, following placement of DiI in medial septum, is remarkably similar to the pattern of cholinergic axons in RSg, as detected by acetylcholinesterase histochemical staining, choline acetyltransferase immunoreactivity, or p75 receptor immunoreactivity; this pattern also is strikingly similar to the band of Aβ immunoreactivity. In animals sustaining early damage to the medial septal nucleus (prior to the advent of Aβ immunoreactivity), the band of Aβ in layer III of RSg does not develop; the corresponding band of cholinergic markers also is eliminated. In older animals (after the appearance of the Aβ immunoreactivity) damage to cholinergic afferents by electrolytic lesions, immunotoxin lesions, or cutting the cingulate bundle, result in a rapid loss of the cholinergic markers and a slower reduction of Aβ immunoreactivity. These results suggest that the septal cholinergic axonal projections transport Aβ or amyloid precursor protein (APP) to layer III of RSg.

Detection of intestinal bifidobacteria and lactobacilli in patients with Hirschsprung’s disease associated enterocolitis

The etiology of Hirschsprung's disease associated enterocolitis (HAEC) is unknown. Previous investigations have suggested that several factors such as dilation of proximal bowel, changes in colonic mucosal defence, and overgrowth of toxigenic bacteria may be related with it. This study was to quantify bifidobacteria and lactobacilli in the feces of Hirschsprung's disease (HD) patients with or without enterocolitis and those of normal children. Fresh stool specimens were collected at the first three days of the admission from 30 HD patients (aged 2 weeks to 2 years) and 15 healthy age-matched non-HD patients in the morning once a day for at least three days. All of them have not been given probiotics or antibiotics at least 7 days before stool collection. Hematoxylin-eosin and acetylcholinesterase histochemical staining on rectal biopsies of patients with HD confirmed the diagnosis of HD in all 30 patients. The 30 HD patients were divided into two groups based on the clinical history of enterocolitis: the HAEC group (n=10) and HD group (n=20). Fecal bifidobacteria and lactobacilli were consecutively quantified by SYBR Green I-based real-time PCR assay. Data were analyzed using SAS v. 12.6 for Windows. All tests were two-tailed, and P values <0.05 were considered statistically significant. The mean levels of bifidobacteria were 7.35+/-0.59, 8.16+/-1.17, and 8.35+/-0.74 in the HAEC, HD and control groups, respectively. The bifidobacteria colonization levels were lower in the HAEC group than in the HD and control groups (P<0.05, P<0.001 respectively). The mean level of lactobacilli in the HAEC (5.51+/-0.65) and HD groups (5.87+/-0.78) was significantly lower than that in the control group (6.39+/-0.56) (P<0.05). But there was no difference in log numbers of lactobacilli between HAEC and HD groups (P>0.05). The scarcity of bifidobacteria and lactobacilli in HAEC patients may result in a decrease in epithelial barrier function and be a predisposing factor in the development of HAEC. This decline suggests that treatment with probiotics or prebiotics may be beneficial in these individuals. Further research will focus on whether probiotics can decrease the incidence of HAEC.