Astroglial and neuronal proteins in cerebrospinal fluid as markers of CNS involvement in Lyme neuroborreliosis

Is Lyme neuroborreliosis, even in its early phase, a parenchymatous disorder in the central nervous system (CNS), and not merely a meningitic process? We quantified cerebrospinal fluid (CSF) levels of four nerve and glial cell marker proteins in Lyme neuroborreliosis patients with pretreatment durations of 7–240 days. AH 23 patients had meningo-radiculitis, and six had objective signs of encephalopathy. Glial fibrillary acidic protein (GFAp) pretreatment levels in CSF, and the light subunit of neurofilament protein (NFL) levels were related to clinical outcome and declined significantly after treatment (P < 0.001 and P < 0.01, respectively). NFL was detectable in 11 out of 22 patients, and pre-and post-treatment NFL levels were associated with the duration of neurological symptoms within 100 days prior to treatment. Neuron-specific enolase (NSE) concentrations also decreased after therapy (P < 0.001), while CSF levels of glial S-100 protein remained unchanged. The pretreatment duration of disease was related to postinfectious sequelae. GFAp, NSE and NFL levels in CSF are unspecific indicators of astroglial and neuronal involvement in CNS disease. The findings in the present study are in agreement with the hypothesis that early and late stages of Lyme neuroborreliosis damage the CNS parenchyma.     

Degradation of glial fibrillary acidic protein protein by a calcium dependent proteinase: an electroblot study

In situ and in vitro degradation of glial fibrillary acidic (GFA) protein in mouse spinal cord was examined with electroblots stained for GFA protein by the peroxidase anti-peroxidase method. Non-degraded, intact GFA protein had a molecular weight of 48 Kdaltons and isoelectric points ranging from pH 5.8 to 6.4. The molecular weights to immunoreactive degradation products ranged from 47 to 28 Kdaltons. All of the degradation products had acid shifted isoelectric points (pH 5.8-5.2). Degradation was prevented by chelating calcium with EGTA. In contrast to in situ degradation, degradation in vitro with 3 mM CaCl₂ occured at a faster rate. The effect of pH and temperature on the degradation process were determined by incubating homogenized spinal cords in 3 mM CaCl₂ solutions varying in pH from 4 to 10 and at 4, 37, and 60°C. The greatest number of immunoreactive bands with the lowest molecular weights occurred at pH 8 and 37 °C. The results suggest that turnover of glial filaments is in part controlled by a calcium dependent proteinase active near neutral pH similar to that postulated for neurofilament turnover.     

Acquisition of glial fibrillary acidic protein-containing fibres by astroglial cells in primary culture is orchestrated by a communicable factor

In a previous study we discovered that primary cultures initiated from the whole brain of 21-day foetal rats contained astroblasts that concertedly acquired glial fibrillary acidic protein (GFAP) fibres. The mechanism of this burst of cytoskeletal differentiation could not be investigated in these cultures because it occurred too quickly (completed within 2 h). We report that cultures initiated from the region of the third ventricle display an extended burst of GFAP acquisition whose rate could be markedly reduced by medium changing. Temporary medium deprivation or the addition of cytosine arabinoside to the growth medium had no effect. Our findings suggest that an as yet uncharacterised communicable factor is involved in the orchestration of cytoskeletal differentiation in culture. This factor may be responsible for synchronising the appearance of GFAP-positive cells in the periventricular regions of the foetal brain.     

Differences between adult and neonatal rats in their astroglial response to spinal injury

Transection of the thoracic spinal cord in adult rats produces an astroglial reaction at the lesion site which spreads gradually to lumbar segments. We compared the spread of gliosis in cordotomized adult and neonatal rats in order to evaluate whether or not maturity of long spinal tracts is a precondition for the genesis of this histopathological reaction. By this experiment, we sought to determine whether spread of gliosis is induced by degeneration of nerve fibers in ascending and descending pathways or results from some more general reaction to injury. The spinal cords of 40 neonatal and 30 young adult rats were transected at T5, and 4 to 60 days later the cervical, thoracic, and lumbar segments were examined immunocytochemically for glial fibrillary acidic protein. In the neonatal rats, there was a moderate gliosis at the lesion site by 7 days; this reaction intensified somewhat during the next 60 days but always remained confined to the site of injury. In contrast, the lesion site of adult rats showed a much more intense gliosis; in those animals the response was maximal by 14 days and was characterized by a gradient of decreasing glial reactivity both rostrally and caudally from the transection site. These results support the hypothesis that the spread of gliosis from spinal lesions results from degeneration of the long ascending and descending fiber tracts.     

Autologous sciatic nerve grafts to the rat spinal cord: Immunofluorescence studies with neurofilament and gliofilament (GFA) antisera

Autologous sciatic nerve grafts to the spinal cord contained many regenerating nerve fibers intensely immunofluorescent with neurofilament antisera. Axonal growth was not confined to the graft but also occurred in the surrounding spinal cord. In this location regenerating nerve fibers were invariably associated with Schwann-like cells. The reverse situation, that is, invasion of the graft by GFA-positive astroglia, also occurred but was a more limited phenomenon involving only a few cells.     

Concerted and predictable acquisition of glial fibrillary acidic protein filaments by a cohort of astroblasts in culture

Primary cultures of the 21 day foetal rat brain contain a cohort of astroblasts that concertedly acquire glial fibrillary acidic protein (GFAP) filaments between the 16th and 18th hour after plating. This burst of cytoskeletal differentiation is not observed in cultures initiated from the 18 day foetal brain and is not effected by the addition of cytosine arabinoside, an inhibitor of glial cell proliferation.     

Astroglial alterations in rat hippocampus during chronic lead exposure.

The present study was performed in order to follow the response of astroglial cells in the rat hippocampus to chronic low-level lead exposure. The experiments combined immunohistochemistry using anti-glial fibrillary acidic protein (GFAP) antibody and conventional transmission electron microscopy (EM). Chronic administration with drinking water [1 g% w/v (subclinical dose) of lead acetate dissolved in distilled water] was started through the mother's milk when pups were 7 days old. Following weaning, experimental offspring were treated for 3 months with the same concentration of adulterated water. The group of intoxicated animals and their controls were sacrificed by perfusion-fixation at 30, 60, and 90 days of exposure. After 60 days of lead treatment, staining of GFAP-positive cells demonstrated an astroglial transformation from the quiescent to the reactive state, characterized by an increase in GFAP. In control rats no changes in GFAP immunostaining were observed. The intensity of the astroglial response was enhanced after 90 days of lead intoxication, showing an increment of GFAP immunoreactivity. Quantification of these changes was made by computerized image analysis, confirming that the sectional areas of the astroglia in lead-exposed animals were larger than those in controls. These results are consistent with the ultrastructural alterations. Simultaneously with the increment in gliofilaments, intranuclear inclusions were seen in some astrocytes. The mechanisms by which lead affects astrocytes are unknown. Probably the astroglial changes induced by lead intoxication produce microenvironmental modifications that may disturb the neuronal function.     

急性脑梗死患者血清胶质纤维酸性蛋白含量的动态变化及其临床意义

目的探讨急性脑梗死(ACI)患者血清胶质纤维酸性蛋白(GFAP)含量的动态变化及其临床意义。方法检测47例ACI患者发病<48h、第3d和第5d的血清GFAP含量,按牛津郡社区卒中项目(OCSP)和CT分型对患者的血清GFAP含量进行比较,并分析其与病情、预后的关系。结果血清GFAP含量在OCSP各亚型患者中,完全前循环梗死(TACI)组发病<48h时明显高于对照组和其他3组,第3d、第5d继续上升;部分前循环梗死(PACI)组在发病第3d达高峰,第5d下降至接近正常水平;而后循环梗死(POCI)组和腔隙性梗死(LACI)组与对照组相比差异无显著性(均P>0.05)。血清GFAP含量在不同面积ACI患者中,发病<48h时各组血清GFAP含量差异无显著性(P>0.05);大面积梗死组第3d、第5d血清GFAP含量明显高于其他4组(均P<0.05),其他4组血清GFAP含量差异均无显著性(均P>0.05)。血清GFAP含量与梗死灶部位无明显相关性,仅第3d的皮质 皮质下组GFAP含量显著高于同时间的其他3组。血清GFAP含量与国立卫生研究所卒中量表(NIHSS)评分呈显著正相关(r=0.410,P<0.01);在病情好转组、未变组及恶化组差异无显著性(均P>0.05),不是预后的独立危险因素。结论ACI后血清GFAP含量的变化可反映病灶大小和病情的严重程度,为指导临床的诊治提供依据。     

Olfactory Schwann cells are derived from precursor cells in the olfactory epithelium

In this morphological and immunohistochemical study we show that olfactory schwann cells (OSC) are derived from precursor cells residing in the olfactory epithelium. During development, they migrate out of the epithelium and extend processes to ensheath the olfactory axons. Olfactory mucosa from E14 rat embryos and juvenile rats were treated with trypsin-pancreatin to remove the underlying connective tissue. The epithelial explant was then maintained for two days in culture, during which cells migrated out from the explant. Among them were spindly bipolar cells which were identified as OSC by their positive immunoreaction for glial fibrillary acidic protein, ultrastructure, and association with growing axons. Axonal growth was significantly more profuse in the embryonic explants, in which the polarity of the OSC was oriented parallel with the axons. Ultrastructural observations showed that ensheathment of the bundles of axons resembled those in vivo.     

Measurement of glial fibrillary acidic protein (GFAP) and anti-GFAP antibodies by solid-phase radioimmunoassays

A solid-phase radioimmunoassay was developed for the detection of glial fibrillary acidic protein (GFAP) and GFAP-specific immunoglobulin G (IgG) antibodies. In antibody assays purified GFAP was adsorbed onto polystyrene beads, followed by incubation in dilutions of serum.
(125-I)-labelled human anti-IgG was used to quantify antibodies bound to solid-phase GFAP. GFAP in samples was measured by the inhibition of the binding of anti-GFAP antibody to solid-phase GFAP.
The serum and CSF samples of 19 brain tumor, 40 multiple sclerosis and 66 control patients were assayed for anti-GFAP antibodies. The binding values in the serum and CSF samples of the brain tumor patient group were higher and the binding values in the CSF samples of the multiple sclerosis group lower than those of the control group. The differences were statistically significant.     

Axotomy-induced neuronal death and reactive astrogliosis in the lateral geniculate nucleus following a lesion of the visual cortex in the rat

Following a unilateral lesion of the visual cortex (cortical areas 17, 18, and 18a) in adult rats, neurons in the ipsilateral dorsal lateral geniculate nucleus (LGN) are axotomized, which leads to their atrophy and death. The time course of this neuronal degeneration was studied quantitatively, and the astroglial response was examined with glial fibrillary acidic protein immunohistochemistry. More than 95% of the neurons in the ipsilateral LGN survive during the first 3 days following a lesion of the visual cortex. However, in the next 4 days, massive neuronal death ensues, reducing the number of surviving neurons to approximately 33% of normal by the end of the first postoperative week. Between 2 weeks and 24 weeks postoperatively, the number of neurons present in the LGN declines very gradually from 34% to 17% of normal. Three days after a lesion of the visual cortex, the mean cross-sectional areas of ipsilateral LGN neurons are 13% smaller than normal (87%). By 1 week after the operation, surviving LGN neurons have atrophied to 66% of their normal area. Subsequently, the size of surviving neurons declines slowly to approximately 50% of normal at 24 weeks after the cortical lesion. Astrocytes in the ipsilateral LGN also react to cortical damage. At 1 day after a lesion of the visual cortex, glial fibrillary acidic protein immunoreactivity in the LGN is almost undetectable, but a distinct increase in immunoreactivity is seen at 3 days. Immunoreactivity peaks between 1 week and 2 weeks postoperatively and, thereafter, remains intense for at least 24 weeks. Thus, following a lesion of the visual cortex, the somata of neurons in the LGN remain essentially normal morphologically for about 3 days before the onset of rapid atrophy and death. Moreover, most of the neural cell death that occurs in the LGN after axotomy takes place in the last half of the first postoperative week. J. Comp. Neurol. 392:252–263, 1998. © 1998 Wiley-Liss, Inc.     

Bone Morphogenetic Proteins: Neurotrophic Roles for Midbrain Dopaminergic Neurons and Implications of Astroglial Cells

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor β (TGF-β) superfamily that have been implicated in tissue growth and remodelling. Recent evidence suggests that several BMPs are expressed in the developing and adult brain. Specifically, we show that BMP 2 and BMP 6 are expressed in the developing midbrain floor of the rat. We studied potential neurotrophic effects of BMPs on the in vitro survival, transmitter uptake and protection against MPP+ toxicity of mesencephalic dopaminergic neurons cultured from the embryonic midbrain floor at embryonic day (E) 14. At 10 ng/ml and under serum-free conditions, most BMPs promoted the survival of dopaminergic neurons visualized by tyrosine hydroxylase immunocytochemistry during an 8-day culture period, but to varying extents (relative potencies: BMP 6 = 12 > 2, 4, 7). BMPs 6 and 12 were as effective as fibroblast growth factor-2 (FGF-2) and glial cell line-derived neurotrophic factor, promoting survival 1.7-fold compared with controls. BMPs 9 and 11 were not effective. Dose-response curves revealed an EC₅₀ for BMPs 2, 6 and 12 of 2 ng/ml. BMPs 2, 4, 6, 7, 9 and 12 also promoted DNA synthesis and astroglial cell differentiation, visualized by 5-bromodeoxyuridine (BrdU) incorporation and glial fibrillary acidic protein (GFAP) immunocytochemistry respectively. Suppression of cell proliferation and subsequent maturation of GFAP-positive cells by 5-fluorodeoxyuridine or aminoadipic acid abolished the neuron survival-promoting effect of BMP 2. This suggests that BMPs, like other non-TGF-β factors affecting dopaminergic neuron survival, act indirectly, probably by stimulating the synthesis and/or release of glial-derived trophic factors. BMP 6 and BMP 7 also increased the uptake of [³H]dopamine without affecting the uptake of [³H]5-hydroxytryptamine and [³H]GABA, underscoring the specificity of the trophic effect. We conclude that several BMPs share a neurotrophic capacity for dopaminergic midbrain neurons with other members of the TGF-β superfamily, but act indirectly, possibly through glial cells.     

Neurofilament light,glial fibrillary acidic protein,and tau in a regional epilepsy cohort: High plasma levels are rare but related to seizures

Objective

Higher levels of biochemical blood markers of brain injury have been described immediately after tonic–clonic seizures and in drug-resistant epilepsy, but the levels of such markers in epilepsy in general have not been well characterized. We analyzed neurofilament light (NfL), glial fibrillary acidic protein (GFAP), and tau in a regional hospital-based epilepsy cohort and investigated what proportion of patients have levels suggesting brain injury, and whether certain epilepsy features are associated with high levels.

Methods

Biomarker levels were measured in 204 patients with an epilepsy diagnosis participating in a prospective regional biobank study, with age and sex distribution correlating closely to that of all patients seen for epilepsy in the health care region. Absolute biomarker levels were assessed between two patient groups: patients reporting seizures within the 2 months preceding inclusion and patients who did not have seizures for more than 1 year. We also assessed the proportion of patients with above-normal levels of NfL.

Results

NfL and GFAP, but not tau, increased with age. Twenty-seven patients had abnormally high levels of NfL. Factors associated with such levels were recent seizures (p = .010) and epileptogenic lesion on radiology (p = .001). Levels of NfL (p = .006) and GFAP (p = .032) were significantly higher in young patients (<65 years) with seizures ≤2 months before inclusion compared to those who reported no seizures for >1 year. NfL and GFAP correlated weakly with the number of days since last seizure (NfL: r_s = −.228, p = .007; GFAP: r_s = −.167, p = .048) in young patients. NfL also correlated weakly with seizure frequency in the last 2 months (r_s = .162, p = .047).

Significance

Most patients with epilepsy do not have biochemical evidence of brain injury. The association with seizures merits further study; future studies should aim for longitudinal sampling and examine whether individual variations in NfL or GFAP levels could reflect seizure activity.     

The relationship between glial distortion and neuronal changes following intestinal ischemia and reperfusion

Background Damage to mucosal epithelial cells, muscle cells and enteric neurons has been extensively studied following intestinal ischemia and reperfusion (I/R). Interestingly, the effects of intestinal I/R on enteric glia remains unexplored, despite knowledge that glia contribute to neuronal maintenance. Here, we describe structural damage to enteric glia and associated changes in distribution and immunoreactivity of the neuronal protein Hu. Methods The mouse small intestine was made ischemic for 3 h and reperfused from 1 to 12 h. Immunohistochemical localisation of glial fibrillary acidic protein (GFAP), Hu and TUNEL were used to evaluate changes. Key Results At all time points glial cells became distorted, which was evident by their altered GFAP immunoreactivity, including an unusual appearance of bright perinuclear GFAP staining and the presence of GFAP globules. The numbers of neurons per ganglion area were significantly fewer in ganglia that contained distorted glia when compared with ganglia that contained glia of normal appearance. The distribution of Hu immunoreactivity was altered at all reperfusion time points. The presence of vacuoles and Hu granules in neurons was evident and an increase in nuclear Hu, relative to cytoplasmic Hu, was observed in ganglia that contained both normal and distorted glial cells. A number of neurons appeared to lose their Hu immunoreactivity, most noticeably in ganglia that contained distorted glial cells. TUNEL reaction occurred in a minority of glial cells and neurons. Conclusions & Inferences Structural damage to gliofilaments occurs following I/R and may be associated with damage to neighboring neurons.     

Disruption of the blood-brain barrier and neuronal cell death in cingulate cortex,dentate gyrus,thalamus, and hypothalamus in a rat model of Gulf-War syndrome

We investigated the effects of a combined exposure to restraint stress and low doses of chemicals pyridostigmine bromide (PB), N, N-diethyl-m-toluamide (DEET), and permethrin in adult male rats, a model of Gulf-War syndrome. Animals were exposed daily to one of the following for 28 days: (i) a combination of stress and chemicals (PB, 1.3 mg/kg/day; DEET, 40 mg/kg/day; and permethrin, 0.13 mg/kg/day); (ii) stress and vehicle; (iii) chemicals alone; and (iv) vehicle alone. All animals were evaluated for: (i) the disruption of the blood-brain barrier (BBB) using intravenous horseradish peroxidase (HRP) injections and endothelial barrier antigen (EBA) immunostaining; (ii) neuronal cell death using H&E staining, silver staining, and glial fibrillary acidic protein (GFAP) immunostaining; and (iii) acetylcholinesterase (AChE) activity and m2-muscarinic acetylcholine receptors (m2-AChR). Animals subjected to stress and chemicals exhibited both disruption of the BBB and neuronal cell death in the cingulate cortex, the dentate gyrus, the thalamus, and the hypothalamus. Other regions of the brain, although they demonstrated some neuronal cell death, did not exhibit disruption of the BBB. The neuropathological changes in the above four brain regions were highly conspicuous and revealed by a large number of HRP-positive neurons (21-40% of total neurons), a decreased EBA immunostaining (42-51% reduction), a decreased number of surviving neurons (27-40% reduction), the presence of dying neurons (4-10% of total neurons), and an increased GFAP immunostaining (45-51% increase). These changes were also associated with decreased forebrain AChE activity and m2-AchR (19-25% reduction). In contrast, in animals exposed to stress and vehicle or chemicals alone, the above indices were mostly comparable to that of animals exposed to vehicle alone. Thus, a combined exposure to stress and low doses of PB, DEET, and permethrin leads to significant brain injury. The various neurological symptoms reported by Gulf-War veterans could be linked to this kind of brain injury incurred during the war.     

血清胶质纤维酸性蛋白和高血压性脑出血的相关性

目的 探讨血清胶质纤维酸性蛋白(GFAP)与高血压脑出血的关系.方法 采用酶联免疫吸附法对68例急性高血压脑出血患者血清GFAP水平进行动态观察,分析其与出血量、出血部位、神经功能缺损程度评分及预后的关系,并与正常对照组比较.结果 高血压脑出血患者发病1～14 d血清GFAP水平明显高于正常对照组(均P<0.001);发病1～14 d血清GFAP水平与出血量正相关(r1=0.375, P<0.05; r3=0.425,P<0.05; r7=0.614, P<0.01;r14=0.532, P<0.01);也与NDS评分正相关(r1=0.476,P<0.05;r7=0.637,P<0.01);与出血部位不相关;发病后血清GFAP＞20 ng/ml的患者预后不佳.结论 高血压性脑出血患者血清GFAP水平明显升高,并且与出血量、病情和预后相关.     

甲基强的松龙对伽玛刀照射后胶质细胞Cx43和GFAP表达的影响

目的 研究甲基强的松龙（MP）对伽玛刀照射后胶质细胞缝隙连接蛋白43（Cx43）和胶质纤维酸蛋白（GFAP）表达的影响。方法体外培养原代星形胶质细胞,经伽玛刀照射（边缘剂量32Gy）并培养36h后随机分为1μg／ml、5μg／ml、10μg／ml、20μg／ml、30μg／ml、40μg/ml组及实验对照组,各组进一步随机分为48h、72h、96h、120h4个亚组。将各组胶质细胞与相应剂量MP共培养,于48、72、96、120h各时间点采用免疫组化方法检测GFAP及Cx43的表达。结果伽玛刀照射后,胶质细胞Cx43表达降低,GFAp表达增高。添加MP后,20μg／ml、30μg／ml组Cx43呈时间依赖性上调;GFAP表达虽呈时间依赖性上升,但其峰值低于实验对照组,峰值出现时间亦晚于实验对照组。结论MP可上凋伽玛刀照射后胶质细胞Cx43的表达,抑制GFAP表达的上升趋势。