Histochemical Reaction For Acetylcholinesterase Research Articles

Konventionelle histologische Diagnostik in der Koloproktologie

With the introduction of immunohistochemical methods, histopathological diagnosis based on formalin fixed, paraffin embedded tissue in coloproctology has substantially improved. In recent years, the routine use of immunohistochemistry for S100, cathepsin D and a picrosirius red staining has proven to be sufficient for the diagnosis of hypoganglionosis of the myenteric plexus and desmosis of the muscularis propria. In some cases, an immunohistochemical reaction for CD 117 is also necessary for the evaluation of Cajal cells. In contrast, in ultrashort Hirschsprung's disease, aganglionosis of the anal ring, aganglionosis of the musculus corrugator cutis ani, and internal sphincter, the histochemical acetylcholinesterase reaction is essential and not replaceable by any immunohistochemical method.Immunohistochemistry, classical histological stains and enzyme histochemistry are complementary histopathological techniques. In contrast to immunohistochemistry, enzyme histochemistry requires native cryostat sections for the assessment of enzyme activity. As a consequence, biopsy performance and transport to pathology departments should be particularly well organized.

Enzymhistochemie des klassischen und des ultrakurzen Morbus Hirschsprung

Hirschsprung's disease is the most important type of gastrointestinal dysmotility in neonatal pathology. Aberrant craniocaudal migration of neural crest stem cells results in an intestinal aganglionic segment of variable length. In 'classical' Hirschsprung's disease (60-75% of cases), the aganglionic segment spans the rectum and sigma. Ultrashort Hirschsprung's disease (5-10%) is restricted to the most distal 3-4 cm or immediate rectoanal transition only. In the normal enteric nervous system, myenteric ganglia modulate the parasympathetic innervation of the sacral roots S2-S4. The absence of myenteric ganglia in Hirschsprung's disease results in massively increased parasympathetic activity with abundant acetylcholine release and pseudo-obstruction in the aganglionic segment. This can be demonstrated in an enzyme histochemical reaction for acetylcholinesterase on frozen sections, which is sufficient to diagnose the classical disease in rectal mucosal biopsies. In ultrashort Hirschsprung's disease, increased acetylcholinesterase activity is demonstrable only in nerve fibres of the muscularis mucosae and submucosa, but not the lamina propria mucosae. Submucosal and myenteric ganglia are physiologically scarce in the most distal rectum; absence of ganglia in a biopsy of the rectoanal transition must not be (wrongly) interpreted as ultrashort Hirschsprung's disease. Therefore, a diagnosis of ultrashort Hirschsprung's disease can be made exclusively using an enzyme histochemical reaction for acetylcholinesterase.

Microphotometric dynamic analysis of the histochemical acetylcholinesterase reaction

Dynamic analysis of the histochemical reaction of Karnovsky-Roots for acetylcholinesterase activity (AChE) is reported. Two methods were used. The first method was videography and densitometric analysis of frames from the film. The second method was direct microphotometric analysis of the reaction dynamics by the plug-method (measurement of average light transmission through a limited area of preparation or image). Special microchambers were used on the stage of an inverted microscope. The results showed the dynamics of final product accumulation in two structures of rat caudate nucleus: AChE-positive neuropil and the AChE-negative myelin bundle during histochemical incubation. Videography and densitometry of the digital images allowed morphologic and microphotometric analysis of changes in tissue sections during incubation, and the dynamic analysis permitted the study of enzyme kinetics in situ. Problems associated with microphotometric analysis of digital images for quantitative histochemical studies of the enzyme activity are discussed.

The accuracy of acetylcholinesterase reaction in rectal suction biopsy in the diagnosis of Hirschsprung’s disease

Suction rectal biopsy with acetylcholinesterase (AChE) histochemistry has been recognized as a reliable method for the diagnosis of Hirschsprung’s disease (HD). This study compares the final pathologic diagnosis made on paraffin embedded material of 68 colectomy specimens with the histochemical AChE reaction from the same patients previously diagnosed as HD by rectal suction biopsy at the Hospital Infantil Pequeno Principe (Curitiba, Brazil) from 1988 to 1999. The group included 58 male and ten female patients with ages ranging from 7 days to 10 years. Thirty-six patients (52.94%) where under 1 year of age at time of surgery. Two of the 68 patients had previous normal histochemical reactions for AChE: one of them resulted a normal ganglionic segment of bowel and the other one was a 15-day-old boy with total colonic aganglionosis, the only false-negative result in this series. Two patients had inconclusive results and because untreatable clinical symptoms also received surgical treatment. One of them resulted a normal ganglionic bowel and the other one was diagnosed as HD. All surgical specimens from the other 64 patients resulted in various extents of aganglionosis presenting prominent nerve trunks in the submucosal and myenteric plexuses, confirming the previous AChE histochemical diagnosis. In three cases there was total colonic aganglionosis. In this study the rectal suction biopsy associated with the histochemical method of AChE, performed days, months or sometimes years before surgery, resulted in a diagnostic accuracy rate of 95.59%, a positive predictive value of 100% and there were no false-positive results.

Comparative quantitative study of the intrinsic cardiac ganglia and neurons in the rat, guinea pig, dog and human as revealed by histochemical staining for acetylcholinesterase

This study was conducted to determine the overall number of intrinsic neurons distributed through-out the entire heart, in which most neurons are located inside of intramural ganglia and are hidden to observers. For this reason, we attempted to ascertain: (1) how the number of neurons located inside of intrinsic cardiac ganglion is related to its area, and (2) whether this relationship is dependent on age and species of animals. Hearts of rats, guinea pigs, dogs and humans were used to examine intramural ganglia stained histochemically for acetylcholinesterase (AChE). The number and parameters of neurons located inside of 104 ganglia were estimated in serial sections. Although the revealed intrinsic cardiac ganglia varied extremely in shape and size, two different types were identified: the globular and plain ones. In the plain ganglia, perikarya of side by side situated neurons were always intensely stained for AChE and, being clearly discernible, they could be reliably counted in any plain ganglia on total heart preparations using a contact microscope. Contrarily, neuron somata in the globular ganglia were densely packed above one another and their perikarya were almost indiscernible for the observer. Counting of neurons located inside of globular ganglia was possible in serial sections only. The largest cardiac ganglia were revealed in dogs, in which some globular ganglia containing up to 2000 neurons occupied more than 1 mm2. In spite of evident species-dependent differences with respect to frequency of large ganglia, the majority of intrinsic cardiac ganglia both in humans and animals were comparatively small, involved approximately 100-200 nerve cells and occupied an area ranging from 0.01 to 0.17 mm2. Overall, the number of neurons located inside of globular ganglion was related to its area (correlation coefficient = 0.82). However, the correlation coefficients between the globular ganglion area and its neuron number were unequal in different species (0.92 in guinea pig; 0.80 in dog; 0.72 in human; and 0.44 in rat) as well as dependent on (1) ganglion size (0.8 for ganglia equal to or larger than 0.17 mm2 and 0.6 for ganglia smaller than 0.17 mm2) and (2) age of specimens (respectively, 0.98 for juvenile and 0.87 for adult dogs; 0.71 for infants and 0.54 for aged human). In all examined animals and humans, the mean measurements of neuron perikarya were similar (on average, 23 microm in width, 32 microm in length, and 615 microm2 in area) and differences between them were statistically insignificant. However, neuron perikarya of adult dogs and aged humans were significantly larger than those revealed in the juvenile dogs and infants, respectively. Based on the data of this study, we concluded that the number of intrinsic cardiac neurons may be approximated in the total heart preparation via counting and measuring of intramural ganglia, contours of which are well-discernible following a histochemical reaction for AChE.

Cholinergic neurons of the pelvic autonomic ganglia and uterus of the female rat: distribution of axons and presence of muscarinic receptors.

Acetylcholine (ACh) stimulates contraction of the uterus and dilates the uterine arterial supply. Uterine cholinergic nerves arise from the paracervical ganglia and were, in the past, characterized based on acetylcholinesterase (AChE) histochemistry. However, the histochemical reaction for acetylcholinesterase provides only indirect evidence of acetylcholine location and is a nonspecific marker for cholinergic nerves. The present study: (1) reevaluated cholinergic neurons of the paracervical ganglia, (2) examined the cholinergic innervation of the uterus by using retrograde axonal tracing and antibodies against molecules specific to cholinergic neurons, choline acetyltransferase and the vesicular acetylcholine transporter, and (3) examined muscarinic receptors in the paracervical ganglia using autoradiography and a radiolabeled agonist. Most ganglionic neurons were choline acetyltransferase- and vesicular acetylcholine transporter-immunoreactive and were apposed by choline acetyltransferase/vesicular acetylcholine transporter-immunoreactive terminals. Retrograde tracing showed that some cholinergic neurons projected axons to the uterus. These nerves formed moderately dense plexuses in the myometrium, cervical smooth muscle and microarterial system of the uterine horns and cervix. Finally, the paracervical ganglia contain muscarinic receptors. These results clearly reveal the cholinergic innervation of the uterus and cervix, a source of these nerves, and demonstrate the muscarinic receptor content of the paracervical ganglia. Cholinergic nerves could play significant roles in the control of uterine myometrium and vasculature.

Recovery of Corneal Nerve Morphology Following Laser in situ Keratomileusis

Morphological changes in the corneal nerves after laser in situ keratomileusis (LASIK) were investigated and the changes were compared with those observed after creation of the keratectomy flap without subsequent photoablation. After creating the hinged flap, a multizone excimer laser photoablation (myopic correction from 6.00 to 6.66 D; diameter 6 mm) was performed on 27 rabbit corneas. Seven of these 27 rabbits received an automated keratectomy without laser photoablation on the fellow eye. A histochemical acetylcholinesterase reaction was used to demonstrate the changes in the morphology of the corneal nerves 3 days, 2.5 and 5 months after the operations. In all specimens the deepest stromal nerve bundles showed normal morphology. Cut nerve trunks were found at the wound margins and at the level of the flap interphase in the stromal bed. At 3 days, both epithelial and basal epithelial/subepithelial nerves were found at the hinge of the flap but the rest of the flap showed a major loss of epithelial, basal epithelial/subepithelial and superficial stromal nerves. A few new regenerating thin nerve fibers were found to emerge from the cut stromal nerve trunks. They appeared to pass the wound margin into the flap area below the epithelium. At 2.5 and 5 months an increasing number of regenerating nerve leashes were observed to emerge from the cut stromal nerve trunks. They appeared to send anastomosing fibers among the neighboring stromal nerves. By this time the epithelial, basal epithelial/subepithelial and anterior stromal innervation had gained an almost normal nerve density and architecture. In the corneas with the flap only, the epithelial innervation was slightly better spared in the center of the flap, and the stromal changes were somewhat less prominent compared with the LASIK corneas.

Recovery of corneal innervation following photorefractive keratoablation.

To study the morphological recovery of rabbit corneal nerves for up to 12 months after photorefractive keratoablation. The histochemical acetylcholinesterase reaction was used to demonstrate morphological features in rabbit corneal nerves at 1 hour, at 6 weeks, and at 3, 6, or 12 months after photorefractive keratoablation. Photorefractive keratoablation ablated the subepithelial nerve plexus, leaving sharply cut stromal nerves. Epithelial innervation was almost completely restored in 3 months, with innervation proceeding from the epithelial nerve plexuses at the wound margins, but the stromal nerves showed abnormal morphological features throughout the study. Abnormally coiled, regenerating axons emerged from the cut stromal nerves. The first of them were observed to penetrate into the epithelium 3 months postoperatively. The acetylcholinesterase reaction of the epithelial cell membranes was weaker in the wound area, except in the specimens that were observed at 12 months. Epithelial innervation was restored relatively quickly; otherwise, neural recovery was slow. The structure and architecture of the stromal nerves was abnormal even in the the specimens that were observed at 12 months. We hypothesize that corneal nerve damage induced by deep photoablations may correlate with the predictability problems and haze.

Choline acetyltransferase in cochlear root neurons

Cochlear root neurons are a distinct group of cells located in the auditory nerve root in small rodents. Their transmitter is still unknown. Some of our preparations showed immunoreactivity of somata of cochlear root neurons with both polyclonal and monoclonal antibodies against choline acetyltransferase (ChAT) which, despite their very weak histochemical reaction for acetylcholinesterase (AChE), suggested that cochlear root neurons might be cholinergic. To test this, we used a radiometric assay to measure ChAT activities of rat auditory nerve root samples containing cochlear root neurons and of adjacent samples not containing them. There was no significant difference between the low mean ChAT activities of these two groups of samples. Thus, cochlear root neurons are not likely to be cholinergic.

Competitive substrate inhibition in the histochemistry of cholinesterase activity in Alzheimer's disease

We used acetylcholine and butyrylcholine to competitively inhibit the cleavage of acetylthiocholine or butyrylthiocholine in plaques and tangles of Alzheimer's disease. Butyrylcholine was much more effective than acetylcholine in reducing the histochemical reaction for acetylcholinesterase not only in neuronal fibers, but also in plaques and tangles. This is in keeping with biochemical data on acetylcholinesterase and supports the existence of true acetylcholinesterase activity within plaques and tangles. However, 2–4 times higher acetylcholine and buturylcholine concentrations were necessary to inhibit the plaque and tangle bound enzyme. Together with the previously reported different pH optima, this suggests that the plaque- and tangle-bound acetylcholinesterase may represent an altered form of this enzyme.

Cholinergic neurons in an association cortex slab chronically isolated from the cat

Small numbers of short- and long-axon cholinergic interneurons were revealed on a slab of association cortex three weeks after (neuronal) isolation from the cat by means of a histochemical acetylcholinesterase reaction. Short-axon neurons are located at layers II–VI and take the form of mainly spindle-shaped medium sized cells with their axons forming synaptic terminals on pyramidal and stellate neurons of the isolated section. Typical positioning of cholinergic terminals on the perikaryon and proximal portions of cholinoceptive neuron dendrites was noted. Pyramidal cholinoceptive cells may be classed as noncholinergic cells, whereas stellate cells may be either cholinergic or noncholinergic. Long-axon cholinergic interneurons of different shapes and sizes are situated at layers I and VI. Neuronal axons located in these layers run within fibers of the first and subcortical layers, establishing intracortical connections beyond the confines of the isolated section.

Cholinergic structures in the cat auditory cortex (area AI)

Plexuses of cholinergic varicose fibers, differing in density in different layers of the neuropil, were found in area AI of the cat's auditory cortex by the histochemical reaction for acetylcholinesterase: Their density was maximal or average in layer I or deeper layers and minimal in layers II and III. Among cells in area AI those which are cholinergic are a few stellate neurons located in layers II–VI. Axons of some neurons terminate on neighboring cells, those of others (some neurons in layer VI) run into the subcortical layer of arcuate association fibers. Cholinergic terminals are located on the bodies and proximal areas of dendrites of neurons most of which do not contain acetylcholinesterase. Choliniceptive neurons of different sizes and shapes are found in all layers of this region of the auditory cortex.

Prenatal and postnatal development of synapses and acetylcholinesterase staining in the dentate gyrus of the rhesus monkey

Morphogenesis, distribution of cholinergic enzyme acetylcholinesterase and synaptogenesis in the dentate gyrus of the rhesus monkey during the pre- and postnatal periods of development were examined using histological, histochemical and ultrastructural methods. The pattern of neuronal differentiation in the dentate gyrus demonstrated distinct superficial-to-deep and lateral-to-medial gradients. The histochemical reaction for acetylcholinesterase was present on gestation day 120 as minimal staining in the supragranular band and in the inner one-third of the dentate molecular layer. At term, the laminar distribution of the enzyme assumed mature pattern although considerable enhancement in staining intensity was achieved postnatally. At term and at 9 months of postnatal age, the most pronounced enzyme activity was found in the supragranular band and in the inner one-third of the molecular layer. Synaptogenesis in the dentate molecular layer was characterized by the early formation of axo-dendritic contacts on dendritic trunks and branches followed by the appearance of synapses on simple and complex spines. Spines were detected infrequently on gestation day 132. On day 148, they ranged in morphology from short stubby protrusions to pedunculated, triangular processes. The majority of the spines exhibited flat postsynaptic surfaces. Complex, synapse-bearing U- and W-shaped spines were observed rarely at this age but appeared more frequently at term and at 15 months of postnatal age. However, at all ages, including 15 months postnatally, synapses on flat-surfaced simple spines predominated. Most synapses were of the asymmetric variety.With certain exceptions, these features of development of the rhesus dentate gyrus resemble the reported patterns of postnatal ontogenesis of this structure in the rat. However, the ingrowth of cholinergic afferents and the major modifications in synapse structure occur prenatally in the rhesus monkey during the second half of the gestation period. This temporal difference between the two species should receive consideration in the planning of neuroplasticity experiments designed to explore lesion-induced adaptations in afferent growth and synaptogenesis in the rhesus dentate gyrus.

Substance P immunoreaction and acetylcholinesterase activity in the cornea and Gasserian ganglion.

Nerves showing acetylcholinesterase (AChE) activity or immunoreactivity for substance P (SP) were demonstrated in the human cornea. AChE-positive fibres were found in stromal nerve trunks from where they penetrated Bowman's membrane and formed a basal epithelial plexus. Intraepithelial terminals arose from this network. SP immunoreactive nerve fibres showed similar architecture but were fewer. Both SP immunoreaction and histochemical AChE reaction were demonstrated consecutively in the same tissue section cut from both human and rabbit cornea. Stromal nerve trunks were found to contain SP immunoreactive and AChE-positive nerve fibres. However, the AChE-positive fibres were much more frequent than those immunoreactive for SP. In the rabbit Gasserian ganglion all neurons showed AChE activity but only some 20% were SP positive. It is concluded that all the sensory trigeminal nerve fibres of the cornea show AChE activity but only a proportion of them contain SP-like material.

Muscle lineage cytoplasm can change the developmental expression in epidermal lineage cells of ascidian embryos

Cytoplasm from muscle lineage blastomeres of an ascidian embryo can cause cells of a nonmuscle lineage to produce larval tail muscle acetylcholinesterase. Muscle cytoplasm was partitioned microsurgically into epidermal lineage blastomeres at the eight-cell stage. Posterior half-embryos (the two B3 cells) of Ascidia nigra were obtained first by separating the anterior and posterior blastomere pairs at the four-cell stage. At third cleavage, the two B3 cells divide into an ectodermal cell pair that gives rise solely to epidermal tissues, and a mesodermal-endodermal blastomere pair from which the tail muscle cells are derived. When the ectodermal and mesendodermal blastomere pairs were isolated from one another by microsurgery and reared as partial embryos, only cells originating from the mesendodermal blastomeres produced a histochemical acetylcholinesterase reaction. Immediately after cleavage of the isolated B3 cells into ectodermal and mesendodermal cell pairs, the cleavage furrows could be made to disappear by pressing firmly on the mesendodermal cells with a microneedle. Repeated up and down pressure with the microneedle at a new position across the mesendodermal cells caused furrows to reestablish in the new position, thereby incorporating mesodermal cytoplasm and increasing the size of the ectodermal cells. The cytoplasmically altered ectodermal blastomere pairs, which became detached from the mesendodermal cells by this microsurgical procedure, continued to divide and were reared to “larval” stages. One-third of these epidermal partial larvae produced patches of cells containing acetylcholinesterase. These results lend further support to the theory that choice of particular differentiation pathways (embryonic determination) in ascidian embryos is mediated by segregation of specific egg cytoplasmic determinants.

The distribution and origin of glutamate decar☐ylase and choline acetyltransferase in ventral pallidum and other basal forebrain regions

The distribution of choline acetyltransferase (ChAT) and glutamate decar☐ylase (GAD), and the histochemical reaction for acetylcholinesterase have been studied in the basal forebrain and globus pallidus of unoperated rats and in rats with an electrolytic lesion of the nucleus accumbens. ChAT was highly concentrated in the substriatal region, the neostriatum and the lateral part of the rostral substantia innominata. The strongest intensity of staining for acetylcholinesterase was found in the substriatal grey and the neostriatum. Very high GAD activity was found in the substantia innominata, being even slightly higher than that in the pars reticulata of the substantia nigra. The lateral preoptic area, the bed nucleus of the stria terminalis and the globus pallidus also showed high activity of GAD. After lesions of the nucleus accumbens the activity of GAD decreased significantly in the substantia innominata and in a restricted part of the rostroventral globus pallidus, but not in the other regions studied. ChAT activity and acetylcholinesterase staining were unaffected in all regions. The results indicate that a dense GABAergic projection originates in the nucleus accumbens and terminates in the rostral substantia innominata and rostroventral part of the globus pallidus. The study gives neurochemical support to the suggestion that nucleus accumbens may be regarded as a ventral part of the neostriatum and that the rostral substantia innominata may be regarded as a ventral part of the glubos pallidus.

Nerve fibers in culture and their interactions with non-neural cells visualized by immunofluorescence.

Cultures of embryonic mouse spinal cord explants, alone or in combination with rat myotubes, were stained by indirect immunofluorescence using antibodies against three structural proteins to: (a) reveal the distribution of these proteins among different cell types, and (b) test the usefulness of antibody staining to reveal the gross morphology of the neurite network in complex cultures. Affinity column purified antibodies were used against chicken gizzard actin, porcine brain tubulin, and skeletal muscle alpha-actinin. Neurites were stained intensely by anti-actin as was the stress fiber pattern of underlying fibroblasts. With anti-tubulin, the staining of neurites was an order of magnitude more intense than the staining of the microtubule pattern of background fibroblasts. Neurite cell bodies and astrocyte-like glia cells were stained with anti-tubulin and their nuclei remained unstained. Anti-tubulin could thus be used to trace even the finest extensions of nerve processes in spinal cord and spinal cord-muscle cultures. Furthermore, it could be combined with the histochemical reaction for acetylcholinesterase (AChE, EC 3.1.1.7) to demonstrate AChE-positive neurons and specialized nerve-muscle contact sites. The staining of neural elements with anti-alpha-actinin was generally much weaker than with anti-actin and anti-tubulin. Neurites were stained only moderately in comparison to myotube Z lines in the same culture. However, a distinct staining of the periphery of dorsal root ganglion cells was observed. Thus, a protein immunologically related to muscle alpha-actinin is present in the nervous system. In myotubes, Z lines were stained intensely with anti-alpha-actinin while I bands were only faintly stained with anti-actin. In isolated myofibrils, both structures were stained intensely with the same antibody preparations.

Growth of diseased human muscle in combined cultures with normal mouse embryonic spinal cord

Normal and diseased human muscle cells have been grown in combined cultures with 12–14 day embryonic mouse spinal cord explants. Nerve endings on myotubes were found by light and scanning electron microscopy, and motor end-plates were identified by a histochemical reaction for acetylcholinesterase. Contracting myotubes, never observed in cultures of human muscle alone, were found in a culture from normal muscle. Histochemical studies demonstrated the presence of strongly and weakly reacting myotubes for both ATPase pH 9.4 and NADH-TR, but could not be related to the development of fibre types. No differences in morphology or histochemical reactions were found between normal and diseased muscle cells in these combined cultures.

Acetyl disulfide, (CH3COS)2, and bis-(thioacetoy) aurate (I) complex, Au(CH3CHOS)2,-histochemical substrates of unusual properties with acetylcholinesterase.

The histochemical reaction for acetylcholinesterase (AChE) employing acetyl disulfide ([CH3COS]2, or AcDiS) as substrate and 0.006 M Pb(NO3)2 as capturing agent was characterized by (1) a sigmoid curve for velocity of hydrolysis versus substrate concentration and (2) a red precipitate (presumably [CH3COSS]2Pb) at sites of AChE at the motor endplates (MEP's) and (from spontaneous hydrolysis) in the supernatant solution. With the addition of 0.03 M thiolacetic acid (TA) to the incubation medium, which by itself produced little or no histochemical staining, the foregoing characteristics were changed to (1) a bell-shaped curve, with the peak at 0.003 M AcDiS, and (2) a black precipitate (presumably PbS) at the MEP's and in the supernatant solution; in addition, the velocity of hydrolysis was increased approximately 100-fold. Comparable differences were obtained with AcDiS as substrate when using ionic Au+ or Au(S2O3)2–3 as the capturing agent. Results are explained on the basis that in the absence of free heavy metal ion ( i.e., with the Pb[TA]2 or Au[S2O3]2–3 complex), AcDiS combines with AChE at both the anionic and esteratic sites, whereas, in the presence of Pb2+ or Au+, attachment of the substrate occurs only at the esteratic site of the enzyme. Results similar to the former type were obtained when the bis-(thioacetoxy) aurate (I) complex (Au[CH3COS]2– or Au[TA]2– served as both substrate and capturing agent. With both the AcDiS-Au(S2O3)2–3 and Au (TA)2– methods, extremely fine localization of AChE was obtained at the MEP's by electron microscopy.