AChE Staining Research Articles

Acetylcholinesterase histochemistry: A very useful technique in the diagnosis of Hirschsprung’s disease

Hirschsprung’s disease (HD) is a common congenital paediatric disorder. The gold standard for its diagnosis is the demonstration of the absence of ganglion cells on H&E stained sections from a spastic segment of the bowel, still at times; conclusive diagnosis of HD is difficult. This study was done to assess the accuracy of rapid Hematoxylin & Eosin (H&E) staining and AChE histochemistry in combination for the diagnosis of HD, especially in frozen section specimens. Samples from 36 clinically suspected HD cases were evaluated for the presence or absence of ganglion cells on H&E staining on both fresh-frozen and paraffin-embedded sections, whereas AChE staining was done on frozen sections only. Of the 36 cases of frozen section specimens from the spastic region of the colon, GCs were absent in 28 cases on H&E staining while positive staining patterns on AChE histochemistry were observed in 29 cases. Formalin-fixed paraffin-embedded sections showed the absence of GC on H&E staining in 28 cases. The sensitivity and specificity of frozen sections with rapid H&E were 77.78% and 81.82% while the sensitivity and specificity of AChE histochemistry were 80.56% and 81.82% respectively in the diagnosis of HD. Application of AChE histochemistry as an ancillary technique on frozen sections specimens of HD in combination with rapid H&E staining is very useful for definitive diagnosis of HD.

Tanshinone Iia Inhibits Megakaryopoiesis in Immune Vasculitis

Introduction Tanshinone IIA, an active component of Danshen (Salvia miltiorrhiza), has been used for centuries to treat hypercoagulation-related diseases, which attributed to its anti-platelet and anti-inflammatory effects. However, the role of Tanshinone IIA in megakaryocytes, the precursor of platelet within the bone marrow, remains unclear. Therefore, the present study established a rabbit model with immune vasculitis to examine the effect of Tanshinone IIA on megakaryopoiesis and to identify the underlying mechanism(s).Methods Immune vasculitis was established in rabbits (3-4 weeks old) by two intravenous injection of 10% bovine serum albumin (2.5 ml/kg) at two-week interval. Those rabbits were randomly treated with Tanshinone IIA (5 mg/kg/d, 7 d, iv) or aspirin (100 mg/kg/d, 7 d, ig). Megakaryocyte count and CFU-MK formation were measured by Wright's and AChE staining, respectively. Human megakaryotic cell lines Meg-01 and CHRF-288-11 were used to examine the effect of Tanshinone IIA on apoptosis by Annexin V-FITC/PI, mitochondrial membrane potential/JC-1 and Caspase-3 activity assays using flow cytometry.ResultsIn rabbits with immune vasculitis, the platelet count, platelet aggregation and the serum levels of inflammatory cytokines IL-1β, TNF-α and IL-6 were significantly increased when compared to their healthy controls. After 7 days of Tanshinone IIA treatment, all these parameters were significantly reduced, with the inhibitions comparable to those caused by aspirin. In addition, the number of megakaryocytes and the formation of CFU-MK were also statistically increased in rabbits with immune vasculitis, which could be significantly reduced by Tanshinone IIA. In vitro, Tanshinone IIA (1, 3, 10 and 30 μg/ml) also significantly inhibited the formation of CFU-MK of bone marrow cells of BALB/c mice (6-10 weeks) in a dose-dependent manner. In human megakaryocytic cell line Meg-01, Tanshinone ⅡA (10 μg/ml, 72 h) induced apoptosis; both early and late apoptotic rates were significantly increased. In another human megakaryocytic cell line CHRF-288-11, Tanshinone ⅡA (10 μg/ml, 72 h) statistically increased the proportion of depolarized cells, from 9.70% to 14.13%, according to mitochondrial membrane potential using JC-1 assay. The expression of active Caspase-3 in CHRF-288-11 was also significantly increased by Tanshinone ⅡA (10 μg/ml, 72 h) from 5.25% to 15.86%.Conclusion The present study shows that Tanshinone IIA ameliorates immune vasculitis by inhibiting megakaryopoiesis and inducing apoptosis of megakaryocytes, which might explain the anti-platelet and anti-inflammatory effects of Tanshinone IIA. DisclosuresNo relevant conflicts of interest to declare.

Synthesis of Inflammatory Cytokines and Expression of Adhesion Molecules in Megakaryocytes

Inflammatory cytokines and platelet adhesion molecules may play a major role in hemostasis and immuno-pathological processes. Hematopoietic cells of myeloid and lymphoid lineages are known to be important sources of a variety of inflammatory cytokines. However, the immunological role of megakaryocytes is not well known. This project studied the involvement of megakaryocytes on immuno-inflammatory processes and the possible mechanism via the adhesion molecules and the synthesis of relevant cytokines. The expression of interleukin (IL)-1 to IL-10, TNF-a, TNF-g and IFN-g in primary megakaryocytes and four megakaryocytic cell lines: Meg-01, Dami, CHRF-288-11 and M-07e were determined by RT-PCR. The effect of IL-1b, IL-3, IL-6 and TPO on megakaryocyte colony formation (CFU-MK) was studied by using a plasma clot culture system. The CFU-MK was confirmed by AchE staining. The expression of adhesion protein CD36 on human platelets, megakaryocytes and megakaryocytic cell lines was also analyzed by using flow cytometry, ELISA and immunocytochemical methods. Our results showed that CD36 was expressed on platelets, megakaryocytes and the four megakaryocytic cell lines. The relative expression level is as follows: platelets > megakaryocytes > Meg-01 > Dami > CHRF-288-11 > M-07e, suggesting that the level of CD36 expression correlates with the degree of maturity of megakaryocytic differentiation. Inflammatory cytokines TNF-a, IL-1b, IL-3 and IL-6 were detected in primary megakaryocytes and all the four megakaryocytic cell lines, suggesting that different stages of megakaryocytes can be as a source of inflammatory cytokines. IL-1b, IL-3 and IL-6, as well as TPO, play a stimulating effect on CFU-MK formation, suggesting that there is an “autocrine” effect on megakaryopoiesis. Megakaryocytes may involve in immuno-inflammatory processes via the synthesis of inflammatory cytokines and platelet adhesion molecules. DisclosuresNo relevant conflicts of interest to declare.

Cytoarchitecture and myeloarchitecture of the entorhinal cortex of the common marmoset monkey (Callithrix jacchus).

The entorhinal cortex (EC) is associated with impaired cognitive function such as in the case of Alzheimer's disease, Parkinson's disease and Huntington's disease. The present study provides a detailed analysis of the cytoarchitectural and myeloarchitectural organization of the EC in the common marmoset Callithrix jacchus. Data were collected using Nissl and fiber stained preparations, supplemented with acetylcholinesterase and parvalbumin immunohistochemistry. The EC layers and subfields in the marmoset seem to be architectonically similar to those that have been proposed in nonhuman primates and humans to date; however, slight differences could be revealed using the present techniques. Throughout its rostrocaudal length, the entorhinal cortex presents a clear six-layered pattern. The entorhinal cortex is divided into six fields, named mainly in accordance to their rostrocaudal and mediolateral positions. At rostral levels, the neurons tend to be organized in patches that are surrounded by large, thick, radially oriented bundles of fibers, and the deep layers are poorly developed. At caudal levels, the divisions are more laminated in appearance. AChE staining at the borders of adjacent fields are consistent with the changes in layering revealed in Nissl-stained sections, of which the lateral regions of the EC display denser AChE staining than that of the medial banks. PV immunoreactivity was found in the labeled somata, dendrites, and axons in all layers and subdivisions. Additionally, we distinguished three subtypes of PV-immunoreactive neurons: multipolar, bipolar and spherical-shaped neurons, based on the shape of the somata and the disposition of the dendrites.

A New Neural Pathway from the Ventral Striatum to the Nucleus Basalis of Meynert with Functional Implication to Learning and Memory

The cholinergic neurons in the nucleus basalis of Meynert (NBM) are among the first group of neurons known to become degenerated in Alzheimer’s disease, and thus the NBM is proposed to be involved in learning and memory. The marginal division (MrD) of the striatum is a newly discovered subdivision at the ventromedial border of the mammalian striatum and is considered to be one part of the ventral striatum involved in learning and memory. The present study provided evidence to support the hypothesis that the MrD and the NBM were structurally connected at cellular and subcellular levels with functional implications in learning and memory. First, when wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP) was stereotaxically injected into the NBM, fusiform neurons in the MrD were retrogradely labeled with WGA-HRP gray-blue particles and some of them were double stained in brown color by AchE staining method. Thus, cholinergic neurons of the MrD were shown to project to the neurons in the NBM. Second, in anterograde tract-tracing experiments where WGA-HRP was injected to the MrD, the labeled WGA-HRP was found to be anterogradely transported in axons from the MrD to the synaptic terminals with dendrites, axons, and perikaryons of the cholinergic neurons in the NBM when observed under an electronic microscope, indicating reciprocal structural connections between the MrD and the NBM. Third, when bilateral lesions of the MrD were injured with kainic acid in rats, degenerative terminals were observed in synapses of the NBM by an electronic microscope and severe learning and memory deficiency was found in these rats by the Y-maze behavioral test. Our results suggest reciprocal cholinergic connections between the MrD of the ventral striatum and the NBM, and implicate a role of the MrD-NBM pathway in learning and memory. The efferent fibers of cholinergic neurons in the NBM mainly project to the cortex, and severe reduction of the cholinergic innervation in the cortex is the common feature of Alzheimer’s patients. The newly discovered cholinergic neural pathway between the MrD of the ventral striatum and the NBM is supposed involved in the memory circuitries of the brain and probably might play a role in the pathogenesis of the Alzheimer’s disease.

Internal anal sphincter achalasia: data from a nationwide survey of allied disorders of Hirschsprung's disease in Japan.

To investigate the incidence and treatment of internal anal sphincter achalasia (IASA) in Japan based on an analysis of data from a nationwide retrospective cohort study of the allied disorders of Hirschsprung's disease. Five cases of definitive IASA were collected from a nationwide retrospective cohort study conducted from 2001 to 2010 and a search of the Japanese literature. Symptoms developed during the neonatal period in two patients, during early childhood in two, and at school age in one. Symptoms included abdominal distension with severe constipation (n=4) and enterocolitis (n=1). Rectocolonography showed megarectum and no narrow segment in most of the patients. All patients were negative for rectosphincteric reflex. The presence of ganglion cells was demonstrated by H&E or AChE staining from rectal mucosal biopsies or resected full-thickness segments. Two patients were treated conservatively, and three were treated surgically by internal anal sphincter myotomy (n=2) or Lynn procedure (n=1), with satisfactory outcomes. IASA is a rare but distinct entity in Japan. Although the clinical features of IASA resemble those of short- and ultrashort-segment HD, characteristic pathological findings include the presence of ganglion cells. The outcomes of both conservative and surgical treatment are good.

Effects of ultrasound-combined microbubbles on hippocampal AchE fibers in rats

ObjectiveTo investigate the protective effect of ultrasound-combined microbubbles on hippocampal acetylcholinesterase (AchE) fibers in rats. MethodsAccording to random digits table, 60 SD rats were divided into two groups, marrow stromal cells (MSCs) intracranial transplantation group and MSCs intracranial transplantation + ultrasonic microbubbles group. Marrow stromal cells were cultivated and isolated in vitro; 12 weeks after transplantation, spatial learning and memorizing abilities of rats were assessed by Morris water maze; AchE staining method was used to observe changes in density and appearance of AchE staining positive fibers in hippocampal CA1 region. ResultsThere was asignificant increase in spatial learning and memorizing abilities of rats in MSCs intracranial transplantation + ultrasonic microbubbles group. Hippocampal AchE staining suggested an increase in the density of AchE staining positive fibers in MSCs intracranial transplantation group; the fibers were regular, intact and dense. Density of hippocampal AchE positive fibers was negatively correlated with the escape latent period and was positively correlated with percentage of the time needed to cross each platform quadrant. ConclusionsBetter promotion of spatial learning and memorizing abilities of rats in MSCs intracranial transplantation + ultrasonic microbubbles group may be related with the protective effect of ultrasound-combined microbubbles on hippocampal acetylcholine fibers.

Intraoperative rapid diagnosis of Hirschsprung's disease

Objective To evaluate the superiorities and disadvantages of reduced nicotinamide adenine dinucleotide dehydrogenase (NADPH-d),acetylcholinesterase (AchE) enzyme histochemistry and hematoxylin and eosin (HE) staining in the intraoperative rapid diagnosis of Hirschprung' s disease (HD),increase the intraoperative diagnostic accuracy and decrease the risks of reoperation.Methods Eighteen children with HD underwent intraoperative full-thickness intestinal biopsies,including 11 boys,and 7 girls,with average age of (8.6 ± 3) months.Frozen sections at three levels (narrow segments,transitional region and dilated segments) were taken,and the extent of aganglionosis was examined by using NADPH-d staining,traditional rapid HE and AchE staining respectively.TUJ-1,a marker of enteric neural cells,was used for identification of NADPH-d positive myenteric plexus and ganglion cells by immunofluorescence staining.Results The positve rate of NADPH-d staining,HE staining and AchE staining for HD was 94.4％ (17/18),83.3％ (15/18),and 88.9％ (16/18),respectively (P ＞ 0.05).Time comsuming in NADPH-d,HE and AchE staining was (7.0 ± 1.7),(23.2 ± 2.9) and (30.9 ± 2.2) min,respectively(P ＜ 0.01).Conclusion The combination of NADPH-d and HE staining,with high idiosyncrasy and sensibility,simple and convenient,can be used as a routine intraoperative diagnosis method to identify HD and detect cutting range of colon.AchE staining was the best technique to demonstrate hypertrophic nerve trunks in the mucosa and submucosa and was well suited to suction rectal biopsy. Key words: Hirschprung's disease ; Intraoperative diagnosis; Pathology ; Colorectal surgery

Medial septal lesion enhances general anesthesia response

Electrolytic lesion of the medial septum, a basal forebrain nucleus that projects to the hippocampus, prolonged the emergence from general anesthesia in rats. Septal lesioned rats required a longer time to recover from a loss of righting reflex (LORR) and a loss of tail-pinch response after injectable (20mg/kg i.p. pentobarbital, 5mg/kg i.v. propofol) or volatile (1.5% halothane, 2% isoflurane) anesthetic. When incremental doses of propofol were given i.p., septal lesioned rats as compared to control rats showed LORR at a lower dose of propofol. Similarly, when the rats were exposed to increasing concentrations of isoflurane, the percent of rats showing LORR was leftward shifted for lesioned rats as compared to control rats. Septal lesioned rats as compared to control rats showed decreased locomotor activity when exposed to 1.5% halothane. Lesion of the medial septum was confirmed by thionin-stained histological sections as well as loss of acetylcholinesterase (AchE) staining in the hippocampus, indicating a depletion of septohippocampal cholinergic afferents. Medial septal lesion resulted in a near complete loss of hippocampal theta rhythm during walking and a general decrease in power of the hippocampal EEG at all frequencies (0–100Hz), during walking or immobility. It is concluded that lesion of medial septum, in part through a loss of septohippocampal cholinergic afferents, increased the anesthesia response to volatile and injectable general anesthetics, during both induction and emergence. It is suggested that the septohippocampal system participates in many components of general anesthesia including hypnosis, immobility, and analgesia.

Immunohistochemical characterization of axonal sprouting in mice

Transgenic manipulation of mouse physiology facilitates the preclinical study of genetic risk factors, neural plasticity, and reactive processes accompanying Alzheimer's disease. Alternatively, entorhinal cortex lesions (ECLs) model pathophysiological denervation and axonal sprouting in rat. Given reports of anatomical differences between the mouse and rat hippocampus, application of the ECL paradigm to transgenic mice first requires comprehensive characterization of axonal sprouting in the wild-type. Adult male C57BL/6 mice sustained unilateral transections of the perforant pathway. Subjects were sacrificed at 1, 4, 10, 18, and 28 days postlesion, and hippocampal sections were stained for AChE, the postsynaptic terminal marker drebrin, and the presynaptic terminal proteins SNAP-25, GAP-43, synapsin, and synaptophysin. To examine synaptic turnover and reinnervation, ipsilateral-to-contralateral staining densities were determined within the dentate molecular layer, and shrinkage-corrected ratios were compared to 28 day-yoked sham cases. At 28 days postlesion, ipsilateral terminal marker densities exhibited significant depression. In contrast, qualitative analyses at earlier time points suggested altered AChE staining patterns and increased SNAP-25 and synapsin immunoreactivity in the inner molecular layer (IML) of the dentate gyrus. C57BL/6 mice exhibit synaptic reorganization following perforant path transections. The IML may provide a key target for evaluation and intervention in ECL mouse models.

EFEK ISOFLAVON FITOESTROGEN DARI EKSTRAK Pueraria lobata TERHADAP MEMORI DAN AKTIVITAS KOLINERGIK DI HIPPOKAMPUS CA1 PADA TIKUS HIPOESTROGEN

Data from retrospective and case-conttrole showed that estrogen replacement therapy (ERT) could prevent ang delayed againstAlzheimer Disease (AD). The using of ERT is still controversial because of their long-term side effect. Isoflavon as a part of phytoestrogenhas potential effect as an alternative substitute for ERT. The aim of this study is to examine the effect of phytoestrogen from Pueraria lobataextract in increasing memory’s function and whether it occur by the increased of cholinergic activity in CA1 hippocampus. Exploration studyperformed by three step. First, determined genistein and daidzein concentration in Pueraria lobata extract, as basic of dose determining.Second, oophorectomy performed to made a hypoestrogenic rat’s model; and third, determined method memory’s function in rats. This studyused twenty five female Rattus novergicus strain Wistar, 10 -11 weeks of age, five rats were normal rats (N) as first group and twenty ratswere hypoestrogenic rats for 2nd, 3rd, 4th, and 5th groups. All of the hypoestrogenic rat were devided into 4 groups, each 5 rats. Hypoestrogenicrats group without Isoflavon Genistein Daidzein supplement (OVX), hypoestrogenic rats group with Isoflavon Genistein Daidzeinsupplement (OVX+IGD) dosage of 15, 30, and 60 mg/kgweight/day each. The IGD was given for 21 days. The Morris Water Morris(MWM) test was performed in 17th, 18th, 19th, 20th, and 21st day. On 22nd day rats were killed. Frozen section of the brain was performedimmediately. Cholinergic activities on CA1 hippocampus were examined with AChE staining. A significant difference was found onmemory’s function and cholinergic activities on CA1 hippocampus between OVX and OVX+IGD groups at all doses (p value = 0,000 ofeach). No significant difference on IGD effect toward brain weight (p=0,936) and pyramidal cell number on CA1 hippocampus (p=0,961).It was suggest that increased memory’s function on OVX+IGD groups were caused by the increased of cholinergic activities on CA1hippocampus.Key word : Isoflavones; phytoestrogen; Genistein; Daidzein; Pueraria lobata; Memory; cholinergic.

Isolated hypoganglionosis: systematic review of a rare intestinal innervation defect

Isolated hypoganglionosis (IH) is rare and resembles Hirschsprung's disease. The diagnosis by suction biopsy is difficult. A full-thickness biopsy is essential. Histological features of IH include sparse, small myenteric ganglia, low acetylcholinesterase activity in the lamina propria, and hypertrophy of muscularis mucosae and circular muscle. This review investigates the epidemiology, symptoms, diagnosis, and outcome of patients with IH. A systematic review, using the keywords "isolated hypoganglionosis" and "intestinal hypoganglionosis" was performed. Publications were reviewed for epidemiology, histological methods, operative procedures, follow-up, and patient's outcome. Eleven publications from 1978 to 2009 reported 92 patients with IH (69 males and 23 females, age 4.85 years), presenting with constipation or enterocolitis. Diagnosis of IH was made by full-thickness biopsy and AChE staining in 91% of patients with additional staining in 82%; 54 patients had bowel resection and pull-through procedures; 11 patients had ileostomy or colostomy, and 2 had sphincter myectomy. Complications reported were enterocolitis, constipation, and residual disease. Seven patients (8%) died due to enterocolitis or short-bowel complications. This review confirms that diagnosis of IH is only possible by histochemical examination of a full-thickness biopsy. Despite advanced age at diagnosis of IH, epidemiological and clinical data are similar to aganglionosis.

Necrotizing Enterocolitis in Full-term Neonates: Is it Aganglionosis?

While NEC is primarily a disease of prematurity, full-term infants account for approximately 10% of cases. The aim of this study was to highlight the association of Hirschsprung's disease and NEC in full-term babies which, as far as we could ascertain, has not been reported before. A retrospective study of all surgically treated neonates presenting with NEC from November 1999 to January 2007 was performed. A total of 20 surgically managed neonates presented with NEC during the study period. Three were excluded from the study because they underwent other treatments. Patients were divided into 2 groups: Group 1 consisted of pre-term neonates with weights below 2400 g and Group 2 consisted of full-term neonates with a weight above 2400 g. Nine out of 17 were in Group 2. Peritoneal drain and proximal diversion ileostomy in the most viable part of intestine was done in the neonatal intensive care unit (NICU). Rectal biopsy was taken from 7 out of 9 in Group 2 and from one presenting with cecal perforation in Group 1. Full thickness open rectal biopsy (H&E staining) was the definitive procedure for diagnosis until 2000, when AChE staining began to be used. Duhamel's pull-through was the procedure of choice for neonates with a positive rectal biopsy. Aganglionosis was found in 4 of 7 rectal biopsies in patients weighing more than 2400 g (Group 2). One died at home while waiting for definitive surgery. Duhamel's pull-through was performed for the rest. Altogether 3 patients died (17.6%). The survivors are developing normally, only one has exhibited retarded growth. One infant suffered from cholestatic jaundice. Our results support the suspicion that a large proportion of term neonates presenting with NEC have long segment or total colonic Hirschsprung's disease. This observation is in contrast to the currently available literature on this problem from the last decade.

Cholinesterase inhibition and acetylcholine accumulation following intracerebral administration of paraoxon in rats

We evaluated the inhibition of striatal cholinesterase activity following intracerebral administration of paraoxon assaying activity either in tissue homogenates ex vivo or by substrate hydrolysis in situ. Artificial cerebrospinal fluid (aCSF) or paraoxon in aCSF was infused unilaterally (0.5 μl/min for 2 h) and ipsilateral and contralateral striata were harvested for ChE assay ex vivo. High paraoxon concentrations were needed to inhibit ipsilateral striatal cholinesterase activity (no inhibition at < 0.1 mM; 27% at 0.1 mM; 79% at 1 mM paraoxon). With 3 mM paraoxon infusion, substantial ChE inhibition was also noted in contralateral striatum. ChE histochemistry generally confirmed these concentration- and side-dependent effects. Microdialysates collected for up to 4 h after paraoxon infusion inhibited ChE activity when added to striatal homogenate, suggesting prolonged efflux of paraoxon. Since paraoxon efflux could complicate acetylcholine analysis, we evaluated the effects of paraoxon (0, 0.03, 0.1, 1, 10 or 100 μM, 1.5 μl/min for 45 min) administered by reverse dialysis through a microdialysis probe. ChE activity was then monitored in situ by perfusing the colorimetric substrate acetylthiocholine through the same probe and measuring product (thiocholine) in dialysates. Concentration-dependent inhibition was noted but reached a plateau of about 70% at 1 μM and higher concentrations. Striatal acetylcholine was below the detection limit at all times with 0.1 μM paraoxon but was transiently elevated (0.5–1.5 h) with 10 μM paraoxon. In vivo paraoxon (0.4 mg/kg, sc) in adult rats elicited about 90% striatal ChE inhibition measured ex vivo, but only about 10% inhibition measured in situ. Histochemical analyses revealed intense AChE and glial fibrillary acidic protein staining near the cannula track, suggesting proliferation of inflammatory cells/glia. The findings suggest that ex vivo and in situ cholinesterase assays can provide very different views into enzyme–inhibitor interactions. Furthermore, the proliferation/migration of cells containing high amounts of cholinesterase just adjacent to a dialysis probe could affect the recovery and thus detection of extracellular acetylcholine in microdialysis studies.

Transplantation of central nervous tissue

Studies of post-lesional reorganization of central nervous connections have shown that central nerve fibers respond to nearby denervation by sprouting and formation of new terminals. The connections in the central nervous system (CNS) are accordingly much more plastic than was thought for a long time. This has revived the interest in transplantation of central nervous tissue. In this study we present some historical data on CNS transplantation supplemented by recent results obtained in our laboratory. Pieces of hippocampal tissue from embryonic or early postnatal rats were transplanted to different parts of the brain of littermates or adult rats. About two-thirds of the transplants were recovered after survival times ranging from 4 d to 2 years, and their cytological organization and intrinsic connections were monitored by cell and fiber stains and histochemical methods (AChE staining and Timm sulphide silver method). Comparison with both a normal and a lesioned control material revealed that in most transplants the tissue had developed as it does when left in situ in the donor brain, but deprived of its major afferent connections. In several instances we found evidence of a major exchange of connections between the transplants and host brains. The conditions needed for this to occur appeared to involve growth stimulation of host brain fibers by transection (host to transplant) and denervation of host neuropil (transplant to host). In cases where these conditions are met, the use of transplants may have future implications in attempts to repair lesions in the central nervous systems.

Hirschsprung’s disease: Is there a relationship between mast cells and nerve fibers?

To define the topography of mast cells and their numbers in cases of Hirschsprung's disease (HD) and non-HD, assess neural hypertrophy using imaging software and to study the relationship between mast cells and nerve fibers. HE stained sections of 32 cases of chronic constipation in the age group of 0-14 years were reviewed for ganglion cells. AChE staining was performed on frozen sections of colonic and rectal biopsies. Based on their findings cases were divided into HD and non-HD and mast cells stained by toluidine blue were evaluated. Image analysis by computerized software was applied to S-100 stained sections for assessment of neural hypertrophy. Difference between number of mast cells in HD group (mean = 36.44) and in non-HD group (mean = 14.79) was statistically significant. Image analysis morphometry on S-100 stained sections served as a useful adjunct. The difference between number, size, and perimeter of the nerve fibers between HD and non-HD group was statistically significant. Mast cells are significantly increased in HD and their base line values are much higher in Indian children than that reported in Western literature. Their role in HD needs further research. Morphometry of S-100 stained nerve fibers is a useful adjunct to conventional methods for diagnosis of HD.

Studies of RET gene expression and acetylcholinesterase activity in a series of sporadic Hirschsprung’s disease

The purpose is to present the studies of RET gene expression and acetylcholinesterase activity in 23 patients operated for Hirschsprung's disease (HD). The patients underwent either transanal endorectal pull-through or Duhamell's procedure. Full-thickness intestinal samples from the three different segments (ganglionic, intermediate and aganglionic) were collected. Each tissue sample was divided in two portions, one for AChE histochemical staining and the other for examination of RET mRNA expression level. All patients had an uneventful postoperative recovery. In all patients, the AChE stainings demonstrated the absence of activity in the ganglionic area, the marked increase of positive fibers in the aganglionic area, and little increase of positive fibers in the intermediate area. In the ganglionic and intermediate areas, all patients (100%) showed significant RET gene expression. In the aganglionic area, 18 patients (78.3%) did not present gene expression and the other five patients (21.7%) presented gene expression that was similar to the ganglionic and intermediate areas. The results reinforce the conclusion that the method of AChE staining is effective for the diagnosis of intestinal aganglionosis and confirm the knowledge that genes beyond RET may be implicated in the genesis of sporadic cases of HD.

Anorectal Electromanometrical Patterns in Children with Isolated Neuronal Intestinal Dysplasia

The aim of this study was to report our new findings on anorectal electromanometrical patterns in patients with isolated neuronal intestinal dysplasia (IND) type B. We reviewed and analyzed the records of preoperative anorectal electromanometric examinations in 17 patients (10 male and 7 female) with IND. The diagnosis of IND was made based on a pathological examination. Mean age of the patients was 6.3 years (range 4 months to 16 years). In the preoperative barium enema study, a narrowed distal segment with proximal dilatation was noted in 8 patients, dilatation of the sigmoid or rectum without a narrowed distal segment was observed in 4 patients, and no specific findings were found in 5 patients. Out of 17 patients, only 5 patients showed positive staining for AChE in the rectal suction specimens. The electromanometric examination showed no significant difference in anal resting pressure and the length of the high pressure zone between the IND and the functional constipation (FC) groups. The frequency of anal peristalsis in the IND group, however, was significantly lower than that in the FC group. Sixteen IND patients showed an internal relaxation. However, the threshold value to evoke the relaxation in the IND group was significantly higher than that in the FC group. Moreover, the latent period of reflex and the duration of a relaxation in the IND group were longer than those in the FC group. Finally, two specific shapes of reflex wave ("W" or "U" shape) were observed in 10 patients with isolated IND type B, while no such shapes were noted in the FC group. These new findings support the notion that anorectal electromanometry is a safe and useful screening examination for IND patients.

Repair of brachial plexus lower trunk injury by transferring brachialis muscle branch of musculocutaneous nerve: anatomic feasibility and clinical trials

There are few effective methods for treating injuries to the lower trunk of brachial plexus, and the curative effect is usually poor. The purpose of this study was to provide anatomic references for transferring the brachialis muscle branch of musculocutaneous nerve (BMBMCN) for selective neurotization of finger flexion in brachial plexus lower trunk injury, and to evaluate its clinical curative effects. Microanatomy and measurement were done on 50 limbs from 25 adult human cadavers to observe the origin, branch, type of the BMBMCN and median nerve, as well as their adjacent structures. Internal topographic features of the fascicular groups of the median nerve at the level of the BMBMCN were observed. In addition, the technique of BMBMCN transfer for selective neurotization of finger flexion of the median nerve was designed and tested in 6 fresh adult human cadavers. Acetylcholinesterase (AchE) staining of the BMBMCN and median nerve was done to observe the features of the nerve fibers. This technique was clinically tried to restore digital flexion in 6 cases of adult brachial plexus lower trunk injury. These cases were followed up for 3, 6, 9 and 12 months postoperatively. Recovery of function, grip strength, nerve electrophysiology and muscle power of the affected limbs were observed and measured. The brachialis muscle was totally innervated by the musculocutaneous nerve (MCN). Based on the Hunter's line, the level of the origin of the BMBMCN was (13.18 +/- 2.77) cm. AchE histochemical staining indicated that the BMBMCN were totally made up of medullated nerve fibers. At the level of the BMBMCN, the median nerve consistently collected into three fascicular groups as shown by microanatomy in combination with AchE stain. The posterior fascicular group was mainly composed of anterior interosseous nerves and branches to the palmaris longus. The technique was tested in six fresh cadavers successfully, except that stoma split occurred in one case. Five of the six cases recovered digital flexion 12 months after operation, and at the same time grip strength, muscle power, and nerve electrophysiology also recovered markedly. The technique of transferring the BMBMCN for selective neurotization of finger flexion is anatomically safe and effective, with satisfactory clinical outcomes.

Botulinium toxin, as bridge to transanal pullthrough in neonate with Hirschsprungs disease

Aims and Objectives:The aim of this study is to find easier way of home care while obviating the colostomy before single stage pull through operation.Materials and Methods:From August 2005 to December 2006, eight cases of neonatal Hirschsprung disease were treated. Mean age 4.5 (2-6) day/old with absent anorectal inhibitory reflex, rectosigmiod disease in Barium enema, positive Acetylcholine esterase (Ache) staining, good response to rectal washout. They underwent botulinium toxin injection (5 unit /kg/quadrant) in four quadrant intrasphincteric. They were followed until pull through operation in 8-10 weeks post injection.Results:Four of 8 (50%) cases only needed rectal washout for three to five days post injection until pull through operation, two had decrease in number of rectal washouts /day and the remaining two underwent colostomy five days post injection because of no response.Conclusion:Botulinium toxin injection can help in palliative care in patients with Hirschsprung disease who are waiting for colostomy or definitive pullthrough. It gives an option of eaiser home care for these patients.