Megacolon in Chagas disease: a study of inflammatory cells, enteric nerves, and glial cells

After acute infestation with the Chagas disease parasite, Trypanosoma cruzi, some patients who are serologically positive develop chronic megacolon and megaesophagus, whereas others are symptom-free. Chagas disease with gastrointestinal involvement involves an inflammatory invasion of the enteric plexuses and degeneration of enteric neurons. It is known that glial cells can be involved in enteric inflammatory responses. The aims were to determine the nature of any difference in lymphocytic invasion, enteric neurons, and enteric glial cells in seropositive individuals with and without megacolon. We have compared colonic tissue from serologically positive individuals with and without symptoms and from seronegative controls. Subjects with megacolon had significantly more CD-57 natural killer cells and TIA-1 cytotoxic lymphocytes within enteric ganglia, but numbers of CD-3 and CD-20 immunoreactive cells were not significantly elevated. The innervation of the muscle was substantially reduced to about 20% in megacolon, but asymptomatic seropositive subjects were not different to seronegative controls. Glial cell loss occurred equally in symptomatic and unaffected seropositive subjects, although the proportion with glial fibrillary acidic protein was greater in seropositive, nonsymptomatic subjects. Development of megacolon after acute infection with T cruzi is associated with maintained invasion of enteric ganglia with cytotoxic T cells and loss of muscle innervation, but changes in glial cell numbers are not associated with progression of enteric neuropathy.     

Enteroglial cells act as antigen-presenting cells in chagasic megacolon

Chagas disease is one of the most serious parasitic diseases of Latin America, with a social and economic impact far outweighing the combined effects of other parasitic diseases such as malaria, leishmaniasis, and schistosomiasis. In the chronic phase of this disease, the destruction of enteric nervous system components leads to megacolon development. Besides neurons, the enteric nervous system is constituted by enteric glial cells, representing an extensive but relatively poorly described population within the gastrointestinal tract. Several lines of evidence suggest that enteric glial cells represent an equivalent of central nervous system astrocytes. Previous data suggest that enteric glia and neurons are active in the enteric nervous system during intestinal inflammatory and immune responses. To evaluate whether these cells act as antigen-presenting cells, we investigated the expression of molecules responsible for activation of T cells, such as HLA-DR complex class II and costimulatory molecules (CD80 and CD86), by neurons and enteric glial cells. Our results indicate that only enteric glial cells of chagasic patients with megacolon express HLA-DR complex class II and costimulatory molecules, and hence they present the attributes necessary to act as antigen-presenting cells.     

Regenerative process evaluation of neuronal subclasses in chagasic patients with megacolon

Chagas’ disease is one of the most serious parasitic diseases of Latin America, with a social and economic impact far outweighing the combined effects of other parasitic diseases such as malaria, leishmaniasis and schistosomiasis. In the chronic phase of this disease, the destruction of enteric nervous system (ENS) components leads to megacolon development. Previous data presented that the regeneration tax in the ENS neurons is augmented in chagasic patients. Although, there are several neuronal types with different functions in the intestine a detailed study about the regeneration of every neuronal type was never performed before. Therefore, the aim of this study was to evaluate the regeneration tax of every neuronal cell type in the ENS from chagasic patients with megacolon and non-infected individuals. A neuronal regeneration marker (GAP-43) was used in combination with a pan-neuronal marker (Peripherin) and several neuropeptides markers (cChat, Substance P, NPY, VIP and NOS), and it was considered as positive just with the combination of these markers. Our results demonstrated that the regeneration levels of cChat, Substance P, and NPY were similar in chagasic patients and non-infected individuals. However, levels of VIP and NOS neuropeptides were increased in chagasic patients when compared with non-infected individuals. We believe that the augment in the regeneration occur due to an increased destruction of selective neuronal types. These results corroborates with previous studies that pointed out to selective destruction of VIP and NOS neurons in chagasic patients.     

Interstitial cells of Cajal in patients with chagasic megacolon originating from a region of old endemicity.

Megacolon is the second most frequent and most important digestive manifestation of Chagas' disease. It is characterized by motor disorders and dilatation of the distal segments of the colon. Several theories have been presented to explain the physiopathology of chagasic megacolon, e.g. the plexus theory. Objective: In the present study the distribution of interstitial cells of Cajal (ICC) was evaluated in colon biopsies from chronic chagasic patients originating from a region of old endemicity for Trypanosoma cruzi and for comparison in subjects with other colon disorders. The chagasic patients had been submitted to colectomy for the investigation of other possible mechanisms underlying the physiopathogenesis of megacolons. Design study: Twenty-two colon biopsies (15 from chagasic patients and 7 from nonchagasic patients) were examined. ICC were identified by immunohistochemistry by using the anti-CD117 antibody. The number of ICC was determined in longitudinal and circular muscle layers and in the myenteric plexus, and the results were analyzed by the Kruskal-Wallis and Student t-tests. Results: A reduced number of ICC was observed in all layers and in the myenteric plexus of patients with chagasic megacolon (P<0.05). Conclusions: We conclude that the physiopathological manifestations observed in the large bowel of chagasic patients originate from alterations that occur in the ICC, which play an important role in the control of gut motility.     

Astroglial response in the excitotoxically lesioned neostriatum and its projection areas in the rat

The anatomical distribution of the astrocytic glial reactions, following ibotenic acid-induced neuronal degeneration of the neostriatum in the rat, has been studied immunohistochemically using an antibody directed against the astrocytic marker, glial fibrillary acidic protein. The acute astroglial response to the excitotoxic lesion, determined 7 days post lesion, was compared with a sham-operated group and a chronic group that had received the excitotoxic lesion 6 months prior to histological evaluation.

Total doses of 16–20 μg ibotenic acid injected unilaterally into the head of the neostriatum caused not only a marked neuronal cell loss but was also accompanied by a large increase in the number and size (about 5 times) of glial fibrillary acidic protein-stained astrocytes throughout the neostriatum by 7 days after lesion. Reactive astrocytes were also observed in the major neostriatal projection areas, the globus pallidus and the substantia nigra pars reticulata, at 7 days post lesion, although no neuronal cell loss could be detected in these regions using regular Cresyl Violet staining. Previous studies of lesions identical to the ones used here have shown that globus pallidus and substantia nigra are deafferented as a result of the neostriatal neuronal degeneration. The reactive astrocytes in the striatal projection areas had a 3–5 times larger size than control astrocytes from the same anatomical region.

In animals that received a larger dose of ibotenic acid into the neostriatum (25 μg), neuronal cell loss was also observed in the neocortex and reactive glial fibrillary acidic-stained astrocytes were found in the entire neocortex of the injected hemisphere. In the chronic group, 6 months after the excitotoxic lesion, the astroglial response was clearly diminished or absent in the major neostriatal projection areas, but was still present within the lesioned neostriatum.

The results suggest that focal neuronal destruction can result in widespread astrocytic glial reactions which follow the anatomical connectivity of the lesioned area. This may have implications for the understanding of the multifocal distribution of glial reactions seen in patients with striatal degeneration as a result of Huntington's disease.     

Immunocytochemistry of neuronal and glial markers in retinoblastoma

Summary An immunocytochemical study of 30 retinoblastomas was carried out using antibodies to neuronal and glial markers. The tumours were found to react with antibodies to neuron-specific enolase (NSE), a marker for neuronal elements, and S-100 and glial fibrillary acidic protein (GFAP), both of which are proteins present in glia. Two distinct cell populations were found within the tumour: the first, composed of anaplastic tumour cells at various stages of differentiation, showed both NSE and S-100 immunoreactivity; the second cell type, which immunostained for S-100 and GFAP, resembled mature glial cells. The results of this study indicate that the retinoblastoma may arise from a pluripotential primitive cell partially retaining neuronal and glial characteristics.     

Nestin immunoreactivity in local neurons of the adult rat striatum after remote cortical injury

Nestin is a marker for the neuronal and glial precursor cells and is expressed in reactive astrocytes after brain injury. Following restricted neocortical injury, we found that cells with neuronal morphology in the adult rat striatum became immunoreactive for both nestin and the neuronal marker, microtubule-associated protein 2 (MAP-2), but not for the astroglial marker, glial fibrillary acidic protein (GFAP). The number of nestin-positive cells transiently increased in the striatum. Continuous administration of 5-bromo-2'-deoxyuridine (BrdU) after cortical injury did not reveal any newly generated neurons in the striatum. Double-labeling fluorescent immunocytochemistry revealed that the nestin-positive striatal cells were also substance-P-positive. These findings suggest that some factors released from the injured cortex may induce nestin immunoreactivity in striatal neurons.     

Reactive changes of retinal astrocytes and Müller glial cells in kainate-induced neuroexcitotoxicity

The aim of this study was to investigate reactive changes of astrocytes and Müller glial cells in rats subjected to kainate treatment, which leads to neuronal degeneration in the ganglion cell layer and the inner border of the inner nuclear layer as confirmed by labelling with Fluoro-Jade B, a marker for degenerating neurons and fibres. Both the astrocytes and the Müller glial cells reacted vigorously to kainate injection as shown by their up-regulated expression of nestin, glial fibrillary acidic protein and glutamine synthetase. A major finding was the induced expression of nestin together with glial fibrillary acidic protein beginning at 1 day post-injection of kainate. The marked nestin expression appeared to be most intense at 1 day and was sustained till 2 weeks as compared with the untreated/normal retina. Western blotting analysis confirmed a marked increase in expression of nestin, glial fibrillary acidic protein and glutamine synthetase as compared with untreated/normal retina. Double labelling study revealed that astrocytes and Müller glial cells expressed the radial glia marker nestin, and incorporated bromodeoxyuridine to re-enter into their cell cycle. The induced expression of these proteins in astrocytes and Müller glial cells indicated an induction of gliotic responses and de-differentiation that may be associated with regenerative efforts after kainate-induced injury. Indeed, with the acquisition of an immature molecular profile as manifested by the induced expression of brain lipid-binding protein and doublecortin in astrocytes and Müller glial cells, the potential of these cells to de-differentiate in retinal neurodegeneration is greatly amplified.     

Enteric glial cells immunoreactive for P2X7 receptor are affected in the ileum following ischemia and reperfusion

The aim of this study was to analyze the effect of ischemia and reperfusion injury (IS) on enteric glial cells (EGCs) and neurons immunoreactive for the P2X7 receptor. Intestinal ischemia was induced by obstructing blood flow in the ileal vessels for 35 min. Afterwards, the vessels were reperfused for 14 days. Tissues were prepared for immunohistochemical labeling of P2X7 receptor, HuC/D (Hu) (pan-neuronal marker) and S100β (glial marker); HuC/D (Hu) and glial fibrillary acidic protein (GFAP, glial marker)/DAPI (nuclear marker); or S100β and GFAP/DAPI. Qualitative and quantitative analyses of colocalization, density, profile area and cell proliferation were performed via fluorescence and confocal laser scanning microscopy. The quantitative analyses revealed that a) neurons and EGCs were immunoreactive for P2X7 receptor; b) the P2X7 receptor immunoreactive cells and Hu immunoreactive neurons were reduced after 0 h and 14 days of reperfusion; c) the S100β and GFAP immunoreactive EGCs were increased; d) the profile area of S100β immunoreactive EGCs was increased by IS; e) few GFAP immunoreactive proliferated at 14 days of reperfusion; f) distinct populations of glial cells can be discerned: S100β⁺/GFAP⁺ cells, S100β⁺/GFAP^– cells and S100β^–/GFAP ⁺ cells; g) histological analysis revealed less alterations in the epithelium cells in the IS groups and h) myeloperoxidase reaction revealed increased of the neutrophils in the lamina propria in the IS groups. This study showed that IS is associated with significant neuronal loss, increase of glial cells and altered purinergic receptor expression and that these changes may contribute to intestinal disorders.     

肠易激综合征早期生活事件模型下的大鼠结肠肠神经可塑性研究

目的从神经可塑性角度探讨肠易激综合征的可能发病机制、临床特征及治疗方法。方法雄性SD大鼠出生后连续13 d每天分离3 h。腹壁撤退反射实验用来检测内脏痛觉过敏。肠神经系统结构可塑性可以通过铺片技术和免疫荧光技术,比较近端结肠神经节(HuD阳性细胞)的大小和数目以及胶质细胞(GFAP)的变化来检测。检测近端结肠多组肌间神经丛和黏膜下神经丛肠神经递质类型(ChAT-、VIP-、nNOS-、calbindin-TrKA-、P75-阳性细胞),分析神经递质的可塑性变化。结果新生期应激可致成年鼠内脏敏感性增高,新生期母婴分离可以诱导肠神经结构改变,导致神经元肥大和增生、胶质细胞与神经元的比例增高。神经递质方面,新生期母婴分离组P75和TrkA表达(黏膜下神经丛、肌间神经丛)较正常组均明显增加。ChAT在肌间神经丛表达明显增加,VIP在黏膜下神经丛表达降低,nNOS在肌间神经丛表达增高,Cabindin表达未见明显异常。结论新生期母婴分离可以引起大鼠结肠肠神经可塑性的长期改变。新生期母婴分离诱导的内脏高敏感性中存在肠神经系统可塑性。早期生活事件是引起成年后肠神经系统可塑性改变的重要原因。     

Noradrenergic-induced expression of c-fos in rat cortex: neuronal localization.

β Adrenoceptors in the rat forebrain have been shown to exist predominantly on astrocytes. Studies were undertaken to determine whether the cellular localization of c-fos expression caused by the activation of brain β receptors would have a similar cellular localization. Double label light and electron microscopic immunohistochemical experiments with a glial (glial fibrillary acidic protein, GFAP) and neuronal marker (neurofilament protein, NFP) were undertaken in rats treated with the adrenergic drug, yohimbine. These studies revealed a predominantly neuronal localization of Fos protein in the cerebral cortex. The latter results indicate that neurons are the postsynaptic noradrenergic target cells in which this immediate early gene is expressed in response to the stimulation of β adrenoceptors. The possible relation of these findings to the glial localization of these receptors is discussed.     

Histogenesis and differentiation potential of central neurocytomas

The central neurocytoma, a recently identified rare supratentorial brain tumor in young adults, is characterized by intraventricular location and a usually benign clinical course. In order to elucidate the histogenesis and differentiation potential of this neoplasm, we have undertaken an immunocytochemical and molecular biological study of four central neurocytomas. It was found by immunocytochemistry and immunoblotting that all tumors express neuron-specific enolase and synaptophysin. Western blots also revealed expression of the synaptic vesicle protein, synapsin I, neurofilament protein and glial fibrillary acidic protein in several neurocytomas which failed to exhibit immunoreactivity to these marker antigens on paraffin sections. Immunocytochemical reactions with antibodies to synaptophysin and glial fibrillary acidic protein on adjacent sections demonstrated coexpression in individual tumor cells. The neuronal form of the pp60src protein-tyrosine kinase, an oncogene-product specifically expressed in central nervous system neurons, was not detectable in two central neurocytomas investigated. N-myc, a proto-oncogene frequently amplified in childhood neuroblastomas, was present as a single copy gene in all central neurocytomas, indicating that amplification of this gene is not involved in the pathogenesis of the central neurocytoma. In accordance with ultrastructural evidence of synaptogenesis, we conclude that the central neurocytoma is a neuroectodermal tumor with consistent commitment for neuronal differentiation. Since these tumors retain a potential for additional glial differentiation, we propose an origin from bipotential progenitor cells in the periventricular matrix, which in the mammalian brain persists throughout adult life.     

Glial organization and chondroitin sulfate proteoglycan expression in the developing thalamus

This study examines the early organization of glial cells, together with the expression of chondroitin sulfate proteoglycans in the developing thalamus of ferrets. Glia were identified with antibodies against vimentin and glial fibrillary acidic protein and the chondroitin sulfate proteoglycans were identified by using an antibody against chondroitin sulfate side chains. Our results reveal three striking features of early thalamic development. First, there is a distinct population of glial fibrillary acidic protein-immunoreactive astrocytes (first seen at E30) that resides in the perireticular thalamic nucleus of the primordial internal capsule. These glial fibrillary acidic protein-immunoreactive astrocytes of the perireticular nucleus are transient and form a conspicuous feature of the early developing forebrain. They are first apparent well before any glial fibrillary acidic protein-immunoreactive astrocytes are seen in other regions of the thalamus (at about P8). Further, unlike in other thalamic regions, these peculiar perireticular astrocytes do not express vimentin before they express glial fibrillary acidic protein. Second, in the reticular thalamic nucleus, the radial glial cells express glial fibrillary acidic protein; they are the only ones to do so in the thalamus during development. The glial fibrillary acidic protein-immunoreactive radial glial cells of the reticular nucleus form a rather distinct band across the developing thalamus at these early stages (E30–P1). Finally, and preceding the expression of glial fibrillary acidic protein, the radial glial cells of the reticular nucleus, unlike those in other thalamic regions, are associated closely with the expression of chondroitin sulfate proteoglycans (E20–E30). Later (after E30), the expression of the chondroitin sulfate proteoglycans in the reticular nucleus declines sharply. The significance of this finding is related to the early organization of the cortico-fugal and cortico-petal pathways.     

Detection of glial fibrillary acidic protein and neurofilaments in the cerebrospinal fluid of patients with neurocysticercosis

Neurocysticercosis (NCC) is an infection caused by Taenia solium larval metacestodes in the central nervous system. The glial fibrillary acidic protein (GFAP) and neurofilaments (NFs) can be used as markers of glial and neuronal damage, respectively. We studied the GFAP and NFs of 68, 160 and 200 kDa in the cerebrospinal fluid (CSF) of patients with NCC by Western blotting. Our results showed that patients with NCC had significantly elevated GFAP levels in the CSF compared with the control, whereas NFs of 68, 160 and 200 kDa were not detected in the CFS of NCC patients. We concluded that GFAP could be used as a marker of glial damage in the CFS of NCC patients.     

Cellular localization of angiotensin type 1 receptor and angiotensinogen mRNAs in the subfornical organ of the rat brain

The cellular localization of angiotensin type 1 receptor (AT 1) and angiotensinogen mRNA expression in the subfornical organ (SFO) of the rat brain has been studied by means of non-radioactive in situ hybridization combined with immunocytochemistry for glial fibrillary acidic protein (GFAP) and Neutral red staining. The AT 1 receptor mRNA expression is shown to be within putative nerve cells without any association with the glial fibrillary acidic protein (GFAP)-immunoreactive (IR) cells. In contrast the angiotensinogen cRNA expression is associated predominantly with GFAP-IR cells. The results demonstrate that a neuronal AT 1 receptor mediates the actions of circulating angiotensin II on the SFO and that the angiotensinogen mRNA is predominantly expressed in the SFO astroglial cells.     

An immunohistochemical study on the distribution of glial fibrillary acidic protein, S-100 protein, neuron-specific enolase, and neurofilament in medulloblastomas

In order to clarify the differentiation of medulloblastomas, the authors studied on the morphological features and immunohistochemical expression of glial fibrillary acidic protein (GFAP), S-100 protein, neuron-specific enolase (NSE), and neurofilament (NF) in 31 medulloblastomas. GFAP was detected only in a small number of tumor cells of 5 medulloblastomas; S-100 protein in both small tumor cells and some so-called spongioblastic cells in 16 medulloblastomas; NSE in the more abundant tumor cells and the matrix in 28 medulloblastomas; NF in a few tumor cells of 12 medulloblastomas; GFAP and NF in 2 medulloblastomas, but each of them in different tumor cells. These results suggest that medulloblastomas have a capacity of differentiation along neuronal and/or glial lines. The conventional morphological markers of differentiation in medulloblastomas such as spongioblastic cells and Homer Wright rosettes were not necessarily compatible with expression of immunohistochemical markers such as GFAP or NF. NSE and S-100 protein seem less valuable markers of differentiation because they were detected in both neuronal and glial elements. But NSE, which was observed in most medulloblastomas, might have a value as a marker for medulloblastomas.     

Immunohistochemical localization of peptidylarginine deiminase in the rat brain

The enzyme peptidylarginine deiminase is responsible for the post-translational modification of certain proteins by catalysing the deimination of arginine residues to citrullines. Recently, peptidylarginine deiminase has been purified from rat skeletal muscle and its primary structure determined by molecular cloning. We have used antibodies raised against this enzyme to examine its distribution in the rat central nervous system. A few discrete neuronal cell groups in the telencephalon were selectively stained. These were the septofimbrial nucleus, the anterior commissural nucleus of the hypothalamus, and the anterodorsal thalamic nucleus. The subcommissural organ was intensely stained; but other circumventricular organs were not. In addition, peptidylarginine deiminase-immunoreactivity was found in a large population of small cells throughout the grey and white matter of the rat brain. Some of these cells contained glial fibrillary acidic protein and could thus be identified as astrocytes. However, in many regions these peptidylarginine deiminase-positive cells appeared distinct from the glial fibrillary acidic protein-positive astrocytes. Light and electron microscopic examination indicated that these cells had the morphology of microglia, however they were not stained by a lectin marker for microglia. These cells often surrounded cerebral capillaries, and sent ramifying processes into the neuropil. Peptidylarginine deiminase has been suggested to be involved in the synthesis of the endothelium-derived relaxing factor nitric oxide from arginine-containing molecules. Thus the distinct peptidylarginine deiminase-positive glial cells surrounding cerebral blood vessels may be in a position to regulate local blood flow in response to neuronal activity.     

脑出血患者血浆胶质纤维酸性蛋白浓度的变化

目的:揭示脑出血患者血浆胶质纤维酸性蛋白浓度的变化,探讨并评价其与疾病预后的相关性。方法:收集脑出血患者和同期健康体检合格者各86例。患者静脉血在脑出血后第1、2、3、5和7d获得,健康体检合格者静脉血在体检时获得,ELISA检测血浆胶质纤维酸性蛋白浓度。结果:脑出血患者血浆胶质纤维酸性蛋白浓度在脑出血后第1d升高,第2d到达高峰,后逐渐下降,均显著高于正常对照组(均P<0.01);与入院时美国国立卫生院神经功能缺损评分呈显著正相关(均P<0.01)。死亡组血浆胶质纤维酸性蛋白浓度均显著高于生存组(均P<0.01)。R0C曲线分析显示,脑出血后第1、2、3、5和7d血浆胶质纤维酸性蛋白浓度预测脑出血1月内死亡均有显著价值(均P<0.01),相比之下,第2d的血浆胶质纤维酸性蛋白浓度对脑出血1月内死亡的预测价值最高。结论:血浆胶质纤维酸性蛋白浓度脑出血后显著升高,与疾病预后显著相关。     

Noradrenergic-induced expression of c-fos in rat cortex: Neuronal localization

β Adrenoceptors in the rat forebrain have been shown to exist predominantly on astrocytes. Studies were undertaken to determine whether the cellular localization of c-fos expression caused by the activation of brain β receptors would have a similar cellular localization. Double label light and electron microscopic immunohistochemical experiments with a glial (glial fibrillary acidic protein, GFAP) and neuronal marker (neurofilament protein, NFP) were undertaken in rats treated with the adrenergic drug, yohimbine. These studies revealed a predominantly neuronal localization of Fos protein in the cerebral cortex. The latter results indicate that neurons are the postsynaptic noradrenergic target cells in which this immediate early gene is expressed in response to the stimulation of β adrenoceptors. The possible relation of these findings to the glial localization of these receptors is discussed.     

麝香多肽体外诱导成年大鼠和人骨髓间充质干细胞定向分化为神经元的研究

目的:观察麝香多肽体外定向诱导成年大鼠和人骨髓间质干细胞(Bonemarrowmesenchymalstemcells,BMMSCs)分化为神经元的能力。方法:采用含100-150mg/L麝香多肽的无血清L-DMEM培养基诱导成年大鼠和人BMMSCs分化为神经元,免疫组化鉴定神经元烯醇化酶(NSE)、神经丝蛋白(NF)、胶质纤维酸性蛋白(GFAP)、神经巢蛋白(nestin)的表达。结果:成年大鼠和人BMMSCs在加入含麝香多肽的无血清L-DMEM培养基诱导后,BMMSC胞体收缩,突起伸出,形似神经元;免疫组化显示诱导出的神经元样细胞NSE、NF、nestin表达阳性,GFAP阴性。结论:麝香多肽可以在体外诱导成年大鼠和人BMMSCs定向分化为神经元。