Characterization of maize chitinase-A, a tough allergenic molecule.

Food allergies are recognized as an increasing health concern. Proteins commonly identified as food allergens tend to have one of about 30 different biochemical activities. This leads to the assumption that food allergens must have specific structural features which causes their allergenicity. But these structural features are not completely understood. Uncovering the structural basis of allergenicity would allow improved diagnosis and therapy of allergies and would provide insights for safer food production. The availability of recombinant food allergens can accelerate their structural analysis and benefit specific studies in allergology. Plant chitinases are an example of food allergenic proteins for which structural analysis of allergenicity has only partially been reported. The recombinant maize chitinase, rChiA, was purified from Pichia pastoris extracellular medium by differential precipitation and cation exchange chromatography. Enzyme activity was evaluated by halo-assays and microcalorimetric procedures. rChiA modeling was performed by a two-step procedure, using the Swiss-Model server and Modeller software. Allergenicity of rChiA was verified by immunoblot assays with sera from allergic subjects. rChiA is active in the hydrolysis of glycol chitin and tetra-N-acetylchitotetraose and maintains its activity at high temperatures (70°C) and low pH (pH 3). The molecule is also reactive with IgE from sera of maize-allergic subjects. rChiA is a valuable molecule for further studies on structure-allergenicity relationships and as a tool for diagnosing allergies.

Predicting early psychiatric readmission with natural language processing of narrative discharge summaries.

The ability to predict psychiatric readmission would facilitate the development of interventions to reduce this risk, a major driver of psychiatric health-care costs. The symptoms or characteristics of illness course necessary to develop reliable predictors are not available in coded billing data, but may be present in narrative electronic health record (EHR) discharge summaries. We identified a cohort of individuals admitted to a psychiatric inpatient unit between 1994 and 2012 with a principal diagnosis of major depressive disorder, and extracted inpatient psychiatric discharge narrative notes. Using these data, we trained a 75-topic Latent Dirichlet Allocation (LDA) model, a form of natural language processing, which identifies groups of words associated with topics discussed in a document collection. The cohort was randomly split to derive a training (70%) and testing (30%) data set, and we trained separate support vector machine models for baseline clinical features alone, baseline features plus common individual words and the above plus topics identified from the 75-topic LDA model. Of 4687 patients with inpatient discharge summaries, 470 were readmitted within 30 days. The 75-topic LDA model included topics linked to psychiatric symptoms (suicide, severe depression, anxiety, trauma, eating/weight and panic) and major depressive disorder comorbidities (infection, postpartum, brain tumor, diarrhea and pulmonary disease). By including LDA topics, prediction of readmission, as measured by area under receiver-operating characteristic curves in the testing data set, was improved from baseline (area under the curve 0.618) to baseline+1000 words (0.682) to baseline+75 topics (0.784). Inclusion of topics derived from narrative notes allows more accurate discrimination of individuals at high risk for psychiatric readmission in this cohort. Topic modeling and related approaches offer the potential to improve prediction using EHRs, if generalizability can be established in other clinical cohorts.

Effect of 4-aminopyridine on gravity dependence and neural integrator function in patients with idiopathic downbeat nystagmus.

Downbeat nystagmus (DBN) is a frequent sign in patients with cerebellar degeneration. It consists of an upward drift of the eye that does not depend on vertical head position (spontaneous drift, SD), a gravity-dependent component (GD), and a gaze-evoked drift reflecting gaze-holding impairment (deficient neural integrator function). The potassium-channel blocker 4-aminopyridine (4-AP) is reported to reduce DBN in patients with cerebellar atrophy but with little or no effect in patients with idiopathic DBN. We prospectively studied the effect of 4-AP on all three components in a large (n = 24) group of the clinically frequent idiopathic DBN. DBN was reduced by 22-31% when the head was off the head erect position. In contrast, there was no effect on vertical gaze-evoked drift. This indicates the therapeutic efficacy of 4-AP not only in patients with cerebellar atrophy but also in idiopathic DBN patients. This beneficial effect, which might be missed when gravity-dependent head positions are not tested, was not related to an improvement of gaze-holding deficit. We suggest it may be related to the restored inhibition of the overacting otolith-ocular reflex.

Parecoxib sodium has opioid-sparing effects in patients undergoing total knee arthroplasty under spinal anaesthesia.

BACKGROUND: This multicentre, double-blind, placebo-controlled study compared the opioid-sparing effectiveness and clinical safety of parecoxib sodium over 48 h, in 195 postoperative patients after routine total knee replacement surgery. METHODS: Elective total primary knee arthroplasty was performed under spinal anaesthesia, with a single dose of spinal bupivacaine 10-20 mg, and intraoperative sedation with midazolam 0.5-1.0 mg i.v., or propofol <6 mg kg(-1)h(-1). Patients were randomized to receive either parecoxib sodium 20 mg twice daily (bd) i.v. (n=65), parecoxib sodium 40 mg bd i.v. (n=67), or placebo (n=63) at the completion of surgery, and after 12, 24, and 36 h. Morphine (1-2 mg) was taken by patient-controlled analgesia or by bolus doses after 30 min. RESULTS: Patients receiving parecoxib sodium 20 mg bd and 40 mg bd consumed 15.6% and 27.8% less morphine at 24 h than patients taking placebo (both P<0.05). Both doses of parecoxib sodium administered with morphine provided significantly greater pain relief than morphine alone from 6 h (P<0.05). A global evaluation of study medication demonstrated a greater level of satisfaction among patients taking parecoxib sodium than those taking placebo. Parecoxib sodium administered in combination with morphine was well tolerated. However, a reduction in opioid-type side-effects was not demonstrated in the parecoxib sodium groups. CONCLUSION: Parecoxib sodium provides opioid-sparing analgesic effects in postoperative patients.

Computational approaches towards the rational design of drug-like compound libraries.

During the practice of combinatorial chemistry, it has been realized that molecular diversity is not the only essential feature in a synthetically feasible library. In addition, it is of utmost importance to enrich potential libraries with those molecules which could be converted to viable drug candidates. Given the enormous number of potentially synthesizable compounds, there is a need to design a subset of true "drug-like" compounds. In addition, a paradigm shift in drug discovery has resulted in the integration of pharmacokinetic and drug development activities into early stages of lead discovery. In particular, in silico filters are being developed and used to help identify and screen out compounds that are unlikely to become drugs. This paper highlights recent computational approaches towards the design of drug-like compound libraries, in particular, the prediction of drug-likeness in a more general sense as well as intestinal absorption through passive transport, the permeation of the blood-brain barrier and recent developments towards identification of potentially metabolically unstable molecules. Current computational tools for library design allow the incorporation of medicinal chemistry knowledge into library planning by a variety of methods, ranging from the use of privileged building blocks and simple counting of structural properties (e.g. number of hydrogen bonding partners) to relatively complex regression or neural network-based models to explain oral bioavailability and other pharmacokinetic properties by structural features. These tools are being incorporated more frequently into drug design according to the "rule-of-five" which refers to simple descriptors correlated to oral drug absorption. Combining experimental knowledge with effective computational filtering and prediction of various aspects of drug-likeness thus facilitates the rapid and cost-effective elimination of poor candidates prior to synthesis and helps focus attention on interesting molecules.

Up-regulation of growth-associated protein 43 mRNA in rat medial septum neurons axotomized by fimbria-fornix transection.

Transection of septohippocampal fibres is widely used to study the response of CNS neurons to axotomy. Septohippocampal projection neurons survive axotomy and selectively up-regulate the transcription factor c-Jun. In the present study we investigated whether these cells concomitantly up-regulate the growth-associated protein-43 (GAP-43), a potential target gene of c-Jun implicated in axonal growth and regeneration. Using in situ hybridization histochemistry (ISHH) it was demonstrated that postlesional c-jun mRNA expression is accompanied by an increased expression of GAP-43 mRNA in the medial septum 3 days following fimbria-fornix transection (FFT). The increase reached a maximum at 7 days and gradually declined thereafter (17 days, 3 weeks). Retrograde prelabeling with Fluoro-Gold followed by axotomy and ISHH revealed that GAP-43 mRNA was up-regulated in septohippocampal projection neurons. Colocalization of GAP-43 mRNA and choline acetyltransferase protein showed that GAP-43 mRNA was expressed by cholinergic medial septal neurons after axotomy. Selective immunolesioning of the cholinergic component of the septohippocampal projection with 192 IgG-saporin followed by FFT demonstrated that GAP-43 mRNA was also synthesized by axotomized GABAergic neurons. These results demonstrate an up-regulation of GAP-43 mRNA in axotomized septohippocampal projection neurons independent of their transmitter phenotype which is closely correlated with c-Jun expression. Because the GAP-43 gene contains an AP-1 site, we hypothesize a c-Jun-driven up-regulation of GAP-43 in lesioned medial septal neurons that may contribute to their survival and regenerative potential following axotomy.

Retrograde tracing with Fluoro-Gold: different methods of tracer detection at the ultrastructural level and neurodegenerative changes of back-filled neurons in long-term studies.

Among the available retrograde fluorescent tracers Fluoro-Gold (FG) is particularly advantageous because it (1) is not only detectable by fluorescence microscopy but also immunocytochemically, resulting in an almost complete staining of the dendritic arbor, (2) is visible in lysosome-like structures allowing for the identification of projection neurons at the ultrastructural level, and (3) remains in the labeled neurons for extended periods of time. Photoconversion and immunostaining for FG, respectively, result in a stable, electron-dense reaction product. Thus, the retrogradely labeled cells can be analyzed quantitatively in the light- and electron microscope for their structural characteristics and input synapses. Long-term studies of back-filled neurons provided evidence for neurotoxic effects of FG in these cells.

Retrograde and anterograde tracing combined with transmitter identification and electron microscopy.

Fiber tracts in the brain are formed by neurochemically heterogeneous neuron populations. To distinguish between the different neurons that contribute to a fiber tract it is necessary to combine anterograde and retrograde tracing techniques with immunocytochemistry. In this article, we describe two techniques which allow for the neurochemical identification of retrogradely labeled neurons and anterogradely labeled axons on the ultrastructural level. The identification of the neurotransmitter identity of retrogradely labeled neurons is achieved by combining retrograde Fluoro-Gold tracing with preembedding immunocytochemistry, while the neurotransmitter identity of anterogradely labeled axons can be revealed by combining anterograde Phaseolus vulgaris-leucoagglutinin (PHAL) tracing and postembedding immunostaining.

Intolerance of osteosynthesis material: evidence of dichromate contact allergy with concomitant oligoclonal T-cell infiltrate and TH1-type cytokine expression in the peri-implantar tissue.

BACKGROUND: We report on a 37-year-old man without history of previous allergic disease who developed an aseptic intolerance reaction to a chromium-cobalt alloy, with local discomfort, loosening, and absence of fracture healing. Both in vivo and in vitro allergoimmunologic diagnostic tests were performed. METHODS: Patch testing was done with a European standard series. Specific serum IgE was measured by CAP-FEIA. In addition to immunohistology (APAAP method), peri-implantar tissue was further analyzed by PCR to determine T-cell-receptor-gamma rearrangement and thus the potential clonal (antigen-driven) T-cell repertoire. The actual tissue mRNA expression for IL-4, IL-6, and IFN-gamma was visualized by RT-PCR. RESULTS: Skin testing gave a delayed-type reaction to dichromate. Specific serum IgE to natural rubber latex and grass pollen was found--but without clinical symptoms. Immunohistology revealed a monocytic and dense T-cell infiltrate. The latter, instead of being random, showed an oligoclonal T-cell receptor rearrangement. In addition, there was TH1-type mediator expression (IL-6 and IFN-gamma, but not IL-4). CONCLUSIONS: Skin test, examination of peri-implantar tissue, and the prompt healing after replacement of the osteosynthesis material suggest an allergic reaction. PCR analysis of peri-implantar tissue can further help to identify and understand allergy-mediated implant intolerance reactions.

Classification, clinical findings and operative treatment of degenerative and posttraumatic shoulder disease: what do we really need to know from an imaging report to establish a treatment strategy?

Successful patient care involves interdisciplinary Cupertino. Images allow an interpretation of a static dimension, but may not be sufficient to develop a surgical treatment strategy, since many shoulder problems have its origin in a dynamic pathology. This article outlines dynamic clinical tests of shoulder pathology, classifies various degenerative and posttraumatic shoulder problems and stresses key facts an imaging report should include in order to plan surgery. This article conveys basic knowledge of those tests and the dynamic background of shoulder pathology. Basic surgical treatment principles are summarised briefly.

Trans catalysis in Tn5 transposition.

Synaptic complexes in prokaryotic transposons occur when transposase monomers bind to each of two specific end-binding sequences and then associate to bring the proteins and the two ends of the transposon together. It is within this complex of proteins and DNA that identical catalytic reactions are carried out by transposase on each of the ends of the transposon. In this study, we perform in vitro transposition reactions by combining the methylated inside end (IE(ME)) biased hyperactive Tn5 transposase, Tnp sC7 version 2.0, and the outside end (OE) biased hyperactive Tn5 transposase, Tnp EK/LP, with plasmid DNA containing a transposon defined by one IE(ME) and one OE. These two proteins cooperate to facilitate double end cleavage of the transposon from the plasmid and conversion into transposition products via strand transfer. When one of the hyperactive Tnps is replaced with a catalytically inactive version containing the mutation EA326 (DDE mutant), the predominant reaction product is a linearized plasmid resulting from single end cleavage. Restriction analysis of these linear products reveals that cleavage is occurring on the end distal to that which is bound by the transposase with an intact active site or in trans. Similar in vitro experiments performed with precut transposons and a supercoiled target plasmid demonstrated that the strand transfer reaction is also facilitated by a trans active DDE motif.

Tn5: A molecular window on transposition.

DNA transposition is an underlying process involved in the remodeling of genomes in all types of organisms. We analyze the multiple steps in cut-and-paste transposition using the bacterial transposon Tn5 as a model. This system is particularly illuminating because of the existence of structural, genetic, and biochemical information regarding the two participating specific macromolecules: the transposase and the 19-bp sequences that define the ends of the transposon. However, most of the insights should be of general interest because of similarities to other transposition-like systems such as HIV-1 DNA integration into the host genome.

[Incidence of joint-specific risk factors in patients with advanced cox- and gonarthroses in the Ulm Osteoarthrosis Study]. / Häufigkeit gelenkspezifischer Risikofaktoren bei Patienten mit fortgeschrittenen Cox- und Gonarthrosen in der Ulmer Osteoarthrose-Studie.

AIM OF STUDY: To determine the prevalence of joint specific risk factors in patients with different patterns of advanced hip and knee osteoarthritis (OA). METHODS: We performed a cross-sectional multicenter study in four orthopaedic hospitals in the southwest of Germany. A detailed medical history (date and nature of trauma, conservative and surgical treatment of congenital or acquired joint disorders known as secondary causes of OA) and radiographic evaluation (sequelae of hip dysplasia, slipped capital femoral epiphysis or other malformations) was obtained in 809 patients with advanced hip (n = 420) or knee (n = 389) osteoarthritis, which required unilateral total joint replacement. According to the presence or absence of joint specific risk factors, patients were classified as having secondary or primary (idiopathic) OA. RESULTS: In 41.7% (25.5%) of patients with hip OA and 33.4% (11.1%) of patients with knee OA some predisposing abnormality of the operated (or contralateral) joint could be observed. In hip OA the underlying pathological conditions were mainly hip dysplasia (25.0% in the operated joint and 14.8% in the non-operated joint) and slipped capital femoral epiphysis (7.1% and 14.8%), while knee OA was most often associated with a history of severe trauma (28.6% and 8.3%) CONCLUSION: While there is a lack of comparable investigations in patients with advanced knee OA, the presented data is somewhat contradictory to earlier reports of the prevalence of identified underlying risk factors in patients with hip OA. The reported differences, however, might be attributed to different methodological approaches and could also resemble recent changes in the multifactorial ethiopathologic concept of OA.

Selective in vivo fluorescence labelling of cholinergic neurons containing p75(NTR) in the rat basal forebrain.

The cholinergic system of the rat basal forebrain is used as a model for the homologous region in humans which is highly susceptible to neuropathological alterations as in Alzheimer's disease. Cholinergic cells in the basal forebrain express the low-affinity neurotrophin receptor p75NTR. This has been utilized for selective immunolesioning of cholinergic neurons after internalization of an immunotoxin composed of anti-p75NTR and the ribosome-inactivating toxin saporin. However, the goal of many studies may be not the lesion, but the identification of cholinergic cells after other experimentally induced alterations in the basal forebrain. Therefore, a novel cholinergic marker was prepared by conjugating the monoclonal antibody 192IgG directed against p75NTR with the bright red fluorochrome carbocyanine 3 (Cy3). Three days after intraventricular injection of Cy3-192IgG the fluorescence microscopic analysis revealed a pattern of Cy3-labelled cells matching the distribution of cholinergic neurons. Apparently the marker was internalized within complexes of p75NTR and Cy3-192IgG which were then retrogradely transported to the cholinergic perikarya of the basal forebrain. In addition to the even labelling of somata, a strong punctate-like Cy3-immunofluorescence was seen in structures resembling lysosomes. The specificity of the in vivo staining was proven by subsequent immunolabelling of choline acetyltransferase (ChAT) with green fluorescent Cy2-tagged secondary antibodies. In the medial septum, the diagonal band and the nucleus basalis only cholinergic neurons were marked by Cy3-192IgG. In parallel experiments, digoxigenylated 192IgG was not detectable within cholinergic basal forebrain neurons after intraventricular injection. Presumably, this modified antibody could not be internalized. On the other hand, digoxigenylated 192IgG was found to be an excellent immunocytochemical marker for p75NTR as shown by double labelling including highly sensitive mouse antibodies directed against ChAT. Based on the present findings, future applications of the apparently non-toxic Cy3-192IgG and other antibodies for fluorescent in vivo and in vitro labelling are discussed.

Influences of educational interventions and adverse news about calcium-channel blockers on first-line prescribing of antihypertensive drugs to elderly people in British Columbia.

BACKGROUND: The way in which dissemination of evidence changes medical practice needs to be better understood. Controversy about calcium-channel blockers (CCB) in the past 3 years has provided a natural experiment, enabling assessment of the impact of media stories, a national warning letter, a teleconference, small group workshops, and newsletters on first-line prescribing of antihypertensive drugs. METHODS: We included all physicians (4403) in British Columbia who prescribed a thiazide diuretic, beta-blocker, inhibitor of angiotensin-converting enzyme (ACE), or CCB as the first antihypertensive agent for 36,507 residents aged 66 years and over, with no previous or concurrent sign of underlying cardiovascular disease. We used a database covering all prescriptions to elderly people to measure the change in proportion of newly treated patients who received each class of drug as first-line therapy. We used a matched cohort design for assessment of the teleconference and workshops, a randomised community design for the newsletters, and time-series analysis for the media impacts. FINDINGS: The proportion of patients who received a CCB as first-line therapy declined gradually from 22% in early 1994 to 15% in late 1996. This proportion was not affected by two waves of adverse news about CCBs in 1995, but fell by 5% for 5 months and by 3% for 1 month after two waves in 1996. The proportion of patients who received either a CCB or an ACE inhibitor as first-line therapy, contrary to guidelines, was still 42% overall in 1996. The workshops and newsletters were followed by shifts from first-line CCB to first-line thiazide prescribing. INTERPRETATION: Changes in prescribing practices occur gradually with the accumulation of small impacts from educational interventions and lay media attention.

Region-specific activation of microglial cells in the rat septal complex following fimbria-fornix transection.

Studies of postlesional microglial activation may gain insight into microglia/neuronal interactions in processes of neurodegeneration. We compared the microglial response after axotomy of septohippocampal projection neurons with that seen after selective immunolesioning of cholinergic septohippocampal neurons with the immunotoxin 192 IgG-saporin. Using the microglial marker isolectin B4 from Griffonia simplicifolia (GSA I-B4), we found striking differences in the microglial response between these two lesion paradigms. Following axotomy of septohippocampal neurons by fimbria-fornix transection (ff-t), there was only a moderate and short-lasting microglial reaction in the medial septum (MS) in the early postlesion period. Prelabeling of septohippocampal neurons with Fluoro-Gold (FG) prior to axotomy revealed the survival of most neurons, and only very rarely were microglial cells observed that had phagocytosed FG-labeled debris. In the lateral septum (LS) containing the degenerating terminals of hippocamposeptal fibers transected by ff-t, a heavy reaction of lectin-labeled activated microglial cells associated with high phagocytotic activity was noticed. Unexpectedly, after a long survival time (6 months) following ff-t, we observed an increase in microglial GSA I-B4 labeling in the MS. In contrast, an inverse pattern of the microglial response, i.e., a strong initial reaction in the MS and very little microglial activation in the LS, was observed after immunolesioning. Our results indicate that the microglial reaction in the MS following ff-t differs substantially from that seen in other models of axotomy.

Cholinergic sprouting in the rat fascia dentata after entorhinal lesion is not linked to early changes in neurotrophin messenger RNA expression.

After unilateral entorhinal cortex lesion cholinergic septohippocampal fibres sprout in the denervated fascia dentata. This process is dependent on neurotrophin changes following the lesion. Thus, there is an up-regulation of nerve growth factor and brain-derived neurotrophic factor messenger RNA expression in the denervated granule cells which is detectable 4 h postlesion and returns to control levels by 24 h. Here, using a competitive polymerase chain reaction and in situ hybridization, a transient neurotropin messenger RNA increase could be demonstrated bilaterally following unilateral electrolytic entorhinal cortex lesion. Treatment of the animals with the N-methyl-D-aspartate receptor antagonist dizocilpine maleate blocked this messenger RNA increase, suggesting an involvement of this receptor type in the neurotrophin changes. However, in spite of this blockade, the typical cholinergic sprouting response as visualized with acetylcholinesterase histochemistry was present in animals four weeks after entorhinal cortex lesion. These data suggest that brief initial changes in neurotrophin messenger RNA expression in dentate granule cells are not responsible for the induction of the cholinergic sprouting. Changes in neurotrophin messenger RNA expression occurring immediately postlesion may be linked to glutamate release from entorhinal terminals resulting from the electrolytic lesion of the projection cells in the entorhinal cortex. We hypothesize that later changes in neurotrophin expression, for example in glial cells, are more likely to be related to the cholinergic sprouting process.

Up-regulation of astrocyte-derived tenascin-C correlates with neurite outgrowth in the rat dentate gyrus after unilateral entorhinal cortex lesion.

The extracellular matrix protein tenascin-C has been implicated in the regulation of axonal growth. Using unilateral entorhinal cortex lesions, which induce a massive sprouting response in the denervated outer molecular layer of the rat fascia dentata, the role of tenascin-C for axonal growth was investigated in vivo. Monoclonal antibodies against the neurite outgrowth and anti-adhesive domains of the molecule were employed. Immunostaining was increased throughout the denervated outer molecular layer by day 2, reached a maximum around day 10, and was back to control levels by four weeks post lesion. Growth cone deflecting as well as neurite outgrowth promoting isoforms of tenascin-C were up-regulated after the lesion. Using electron microscopy, single intensely tenascin-C immunoreactive cells were identified as reactive astrocytes that phagocytose degenerated terminals. In situ hybridization histochemistry for tenascin-C messenger RNA revealed numerous cellular profiles in the denervated outer molecular layer of the ipsilateral and contralateral dentate gyrus two days post lesion. Tenascin-C messenger RNA-positive cells in the outer molecular layer were identified as astrocytes using double-labelling for tenascin-C messenger RNA and glial fibrillary acidic protein immunohistochemistry. Thus, a tenascin-C-rich substrate is present in the outer molecular layer during the time of sprouting and a sharp boundary is formed against the inner molecular layer. This pattern may contribute to the layer-specific sprouting response of surviving afferents after entorhinal lesion. Neurite outgrowth may be promoted within the denervated zone, whereas axons trying to grow into the denervated outer molecular layer, for example from the inner molecular layer, would be deflected by a tenascin-C-rich barrier.

Recovery of ChAT immunoreactivity in axotomized rat cholinergic septal neurons despite reduced NGF receptor expression.

Previous studies have suggested that target-derived nerve growth factor (NGF) is essential for the survival of cholinergic basal forebrain neurons. Thus, axotomy of septohippocampal neurons in adult rats resulting in the withdrawal of target-derived NGF caused a dramatic loss of choline acetyltransferase (ChAT)-immunoreactive neurons in the medial septum-diagonal band complex. We have recently shown that this loss of immunolabelled neurons does not indicate cell death, since many septohippocampal cholinergic neurons recover their immunoreactivity for ChAT after a long survival time despite disconnection from target-derived neurotrophins. One possibility would be that these surviving ChAT-immunoreactive neurons have gained access to other, probably local, NGF sources. Here we provide evidence that the recovery of ChAT immunoreactivity after axotomy is not accompanied by a similar recovery of NGF receptor expression in these neurons. In situ hybridization for p75NTR mRNA and trkA mRNA 6 months after bilateral fimbria-fornix transection revealed a substantial loss of labelled cells. In addition, there was a persisting loss of p75NTR-immunoreactive and NGF-immunoreactive medial septal neurons. Cholinergic neurons in controls did not express NGF mRNA, but were heavily immunostained for NGF protein due to receptor-mediated uptake. These data suggest that at least some cholinergic septohippocampal neurons re-express ChAT either independently of NGF or with a reduced need for NGF.

192 IgG-saporin-induced loss of cholinergic neurons in the septum abolishes cholinergic sprouting after unilateral entorhinal lesion in the rat.

After unilateral lesion of the entorhinal cortex, cholinergic septohippocampal fibres are believed to sprout in the denervated outer molecular layer of the rat dentate gyrus. This cholinergic sprouting has been demonstrated by acetylcholinesterase (AChE) histochemistry, a method said selectively to label cholinergic septohippocampal fibres in the hippocampus. However, a recent report has questioned this concept, suggesting that AChE may not be an adequate marker to monitor cholinergic sprouting and that other, non-cholinergic axons sprouting after entorhinal cortex lesion cause the dense AChE-positive band in the denervated outer molecular layer. In order to determine the contribution of cholinergic septohippocampal fibres to the dense AChE band appearing after entorhinal cortex lesion, the neurotoxin 192 IgG-saporin, known to destroy cholinergic neurons in the basal forebrain selectively, was used. Rats received bilateral injections of 192 IgG-saporin into the lateral ventricles 3 weeks before entorhinal cortex lesion, simultaneously with entorhinal cortex lesion, or 8 weeks after entorhinal cortex lesion. Immunocytochemistry for choline acetyltransferase (ChAT) and in situ hybridization for ChAT mRNA demonstrated the loss of cholinergic neurons in the medial septum and diagonal band after 192 IgG-saporin treatment. The cholinergic sprouting response in the molecular layer, as visualized with AChE histochemistry, was abolished in all animals treated with immunotoxin. These data indicate that the dense AChE band forming after entorhinal cortex lesion represents the sprouting of cholinergic septohippocampal fibres.