Bielschowsky Staining Research Articles

Ameliorative effects of aqueous extract of Colocasia esculenta leaf against lipopolysaccharide-induced prefrontal cortex damage in mice

IntroductionThe prefrontal cortex (PFC), crucial for cognition and memory, occupies a third of the cerebral cortex. This study investigated the protective effects of aqueous extract of Colocasia esculenta leaf (AECEL) against lipopolysaccharide (LPS)-induced PFC damage in mice.Methods42 male Swiss albino mice (18–22 g) were divided into six groups. Group A: healthy controls. Groups B-F received daily intraperitoneal LPS injections (0.5 mg/kg) for seven days. Group B: received distilled water, (2 ml) subsequently. Groups C-E: AECEL at 400, 600, and 800 mg/kg, respectively. Group F: donepezil (DPZ, 2.5 mg/kg) by oral gavage. All treatments continued for 28 days.ResultsLPS exposure significantly increased brain oxidative stress markers and reduced antioxidant enzymes compared to controls (p < 0.05). AECEL and DPZ administration significantly reduced brain oxidative stress markers and reduced antioxidant enzymes, as well as tumor necrosis factor-α and interleukin-6 levels (p < 0.05) compared to LPS ONLY group. Y-maze testing revealed decreased alternation (%) in the LPS group compared to controls (p < 0.05). Based on Y-maze performance, AECEL at 400 mg/kg emerged as the most effective dose, significantly improving alternation (%) compared to both LPS and LPS + DPZ groups (p < 0.05). Bielschowsky staining showed senile plaques and neurofibrillary tangles in the LPS group.ConclusionLPS induced PFC damage. Among the tested doses, AECEL at 600 mg/kg demonstrated the most effective improvement in cognitive function, memory, and anxiety-like behaviors as measured by the Y-maze test.

Anabolic Androgenic Steroid predisposes to the neuropathology of Alzheimer’s disease: insights from Animal model

AbstractBackgroundAnabolic androgenic steroids (AASs) are synthetic derivatives of testosterone use for body enhancement by athletes and its use has been abused over the years. Various studies have reported the harmful potential of AAS use, however, there is need to document its possibility as a risk factor in neuropathology of dementia. This study is to demonstrate potential of AAS as a risk factor in Alzheimer’s disease (AD) pathology by demonstrating changes in the neuro‐morphology, astrocytic proliferation and neurofibrillary tangle formation in animal model.MethodTwenty (20) adult male Wistar rats used were divided into four groups (A‐D), treated as follows, Control (A), Olive oil as vehicle (B), oral 120mg/kg of AAS for 21days (C), and 7 days withdrawal group following 120mg/kg/ 21days of AAS intake (D). Serum was assayed for lipid peroxidation marker Malondialdehyde (MDA) and antioxidant enzyme ‐superoxide Dismutase (SOD). The prefrontal cortices were processed and stained for Nissl (chromatophilic) bodies, astrocytes immunohistochemistry and Bielschowsky stain for neurofibrillary tangles.ResultAAS treated group has characteristic pathology of AD demonstrated by the presence of necrosis, chromatolysis, marked astrocytic proliferation and presence of neurofibrillary tangles using Bielschowsky stain along with an increase in MDA and decline in endogenous SOD activities as compared with the control and olive oil treated groups. The withdrawal group (D) had a notable or mild restoration of the neurons demonstrated by presence of few necrotic neurons, reduced reactive astrocytes (astrogliosis) proliferation, decline in MDA with an increase in SOD when compared to the AAS treated group.ConclusionAAS has the potential to predispose individual to the neuropathology of AD characterized by neurodegeneration, astrogliosis, neurofibrillary tangles even in the presence of endogenous antioxidant enzyme, however, upon discontinuation endogenous antioxidant enzyme level increases enabling neuro repair via mobbing off free radicals generated by AAS.

Crosstalk between tau and alpha7 nicotinic acetylcholine receptors at the synapse

AbstractBackgroundThe failure of cholinergic transmission and the increase of amyloid‐β peptide (Aβ) and tau protein are key players in Alzheimer’s disease (AD). The interaction between α7 nicotinic acetylcholine receptors (α7‐nAChRs) and Aβ has been previously investigated, but few studies have focused on the crosstalk between α7‐nAChRs and tau. We previously reported that Aβ and tau might act independently or concomitantly to impair synaptic plasticity and memory, converging onto common targets such as amyloid precursor protein (APP). Here we investigated the role of α7‐nAChRs on tau‐mediated impairment of synaptic plasticity and memory, tau neuropathology, and their interplay with APP.MethodWe used WT (C57Bl6/J) and α7 KO at different ages. 3XTg models of AD were used as positive controls. We performed electrophysiology to investigate synaptic transmission and plasticity at the CA3‐CA1 synapses from hippocampal slices, and behavioral studies (Novel Object Recognition and Location, Fear Conditioning) to evaluate memory. We used western blot for the expression of APP, tau (Ser199, Ser396, Thr205) and GSK‐3β (Ser9) phosphorylation. Formation of tau paired helical filaments (PHFs) and neurofibrillary tangles (NFTs) were evaluated by immunohistochemistry and Bielschowsky staining. Immunoreactivity for NeuN was measured to assess neuronal loss.ResultWe found that α7 KO mice presented an AD phenotype characterized by the impairment of Long‐Term Potentiation and Paired Pulse Facilitation, and the reduction of different types of memory starting at 12 months of age. This age‐dependent phenotype was paralleled by an increase of APP expression, hyperphosphorylation of tau, and a decrease of GSK‐3β (Ser9) phosphorylation. α7 KO hippocampi presented a marked tau neuropathology with an increase of PHF‐1 immunoreactivity, the presence of PHFs and NFTs, and neuronal loss.ConclusionIn summary, the lack of α7‐nAChRs was sufficient to trigger an AD‐like phenotype characterized by a marked tau pathology. Our data also suggest that the concomitant increase of APP expression might contribute to tau neurotoxicity.

Mesoscopic Assessment of Microstructure in Glioblastomas and Metastases by Merging Advanced Diffusion Imaging with Immunohistopathology.

Glioblastomas and metastases are the most common malignant intra-axial brain tumors in adults and can be difficult to distinguish on conventional MR imaging due to similar imaging features. We used advanced diffusion techniques and structural histopathology to distinguish these tumor entities on the basis of microstructural axonal and fibrillar signatures in the contrast-enhancing tumor component. Contrast-enhancing tumor components were analyzed in 22 glioblastomas and 21 brain metastases on 3T MR imaging using DTI-fractional anisotropy, neurite orientation dispersion and density imaging-orientation dispersion, and diffusion microstructural imaging-micro-fractional anisotropy. Available histopathologic specimens (10 glioblastomas and 9 metastases) were assessed for the presence of axonal structures and scored using 4-level scales for Bielschowsky staining (0: no axonal structures, 1: minimal axonal fragments preserved, 2: decreased axonal density, 3: no axonal loss) and glial fibrillary acid protein expression (0: no glial fibrillary acid protein positivity, 1: limited expression, 2: equivalent to surrounding parenchyma, 3: increased expression). When we compared glioblastomas and metastases, fractional anisotropy was significantly increased and orientation dispersion was decreased in glioblastomas (each P < .001), with a significant shift toward increased glial fibrillary acid protein and Bielschowsky scores. Positive associations of fractional anisotropy and negative associations of orientation dispersion with glial fibrillary acid protein and Bielschowsky scores were revealed, whereas no association between micro-fractional anisotropy with glial fibrillary acid protein and Bielschowsky scores was detected. Receiver operating characteristic curves revealed high predictive values of both fractional anisotropy (area under the curve = 0.8463) and orientation dispersion (area under the curve = 0.8398) regarding the presence of a glioblastoma. Diffusion imaging fractional anisotropy and orientation dispersion metrics correlated with histopathologic markers of directionality and may serve as imaging biomarkers in contrast-enhancing tumor components.

Neuropathological autopsy findings in an individual with Alzheimer’s disease who received long‐term treatment with lecanemab (BAN2401)

AbstractBackgroundAnti‐amyloid antibodies administered to patients with Alzheimer’s disease can result in marked reduction of brain amyloid as measured by neuroimaging with amyloid PET ligands. However, there is a lack of pathological data. Here we present neuropathological findings in a patient who received ∼173 weeks of (interrupted) treatment with lecanemab (BAN2401), a humanized IgG1 monoclonal antibody that preferentially targets soluble aggregated β‐amyloid species (protofibrils) and insoluble fibrils, in a Phase 2 study (NCT01767311, Alz Res Therapy 2021;13:80).MethodBrain autopsy was performed on an ∼85 year old whose lecanemab treatment history over 6 years was: 79 weeks 10mg/kg monthly in the double‐blind (DB) phase, then 98 weeks without treatment, then 94 weeks at 10mg/kg biweekly in the open‐label extension (OLE) phase, followed by ∼12 weeks of treatment discontinuation prior to death.ResultClinically, memory symptoms started age ∼77. Study entry was at age ∼80, with mild dementia, CDR 0.5, CDR‐SB 3, and MMSE 24. Florbetapir PET scan was positive at baseline, and negative after 53 weeks DB treatment and at start of OLE. These findings and clinical measures are shown in Table 1. ARIA was not observed during the study. Autopsy showed low brain weight (1052g) with diffuse atrophy, including hippocampi, consistent with atrophy seen on clinical MRI scans. Microscopic findings showed tau pathology including tangles and threads (by AT8 antibody and Bielschowsky staining) throughout neocortex (Braak NF stage VI; B3). Beta‐amyloid immunohistochemical stain (6E10) showed only modest amyloid load (Thal phase 2; A1), with near‐absence of diffuse amyloid deposits and only scattered clusters of neuritic plaques (CERAD stage B; C2).ConclusionIn this autopsy of an individual with Alzheimer’s disease treated with lecanemab, neuropathological changes showed tau and β‐amyloid deposits. However, the pattern of significant tau pathology, accompanied by only a mild degree of β‐amyloid deposits, (A1B3C2), is uncommon in typical (untreated) Alzheimer’s disease. For example, in the NACC neuropathology dataset, only 2% of brains with Braak B2 or B3 show Thal stage A0 or A1. This pattern suggests that lecanemab treatment resulted in a reduction in brain amyloid, consistent with the amyloid PET scanning results in this patient.

Evaluation of Memory Impairment Following Prenatal Exposure to Mosquito Coil Smoke

Background: Developmental Neurotoxicity can lead to the buildup of reactive oxygen species which is an indicator to oxidative stress in the prenatally exposed offspring. Neuronal oxidative stress induces neuroinflammation, precedes tangle formation, and disrupts synaptic plasticity. The result of such changes may be expressed into adulthood as behavioral deficits. All together, these mechanisms are implicated in memory disorders. Objectives: To investigate the histochemical changes in the hippocampus and entorhinal cortex of Wistar rats' offspring after prenatal exposure to mosquito coil smoke and its effect on memory. . Methods: 12 pregnant Wistar rats were grouped into four, 3 animals per group. Group I was exposed to fresh air. Groups II, III, and IV were exposed to mosquito coil smoke for 4, 6 and 8 hours daily respectively throughout gestation period. On Post-natal day (PND) 28 and 29, shortterm spatial and recognition memory of adolescent wistar rats were assessed using water licking task and novel object recognition test respectively. For each animal group (I-IV), a total of 8 animals were randomly selected from the litters for neurobehavioral studies. Experimental animals were humanely sacrificed and sections from the hippocampus and entorhinal cortex were processed for histochemical studies using Bielschowsky stain. Data were presented as mean ± SEM; analysed using One-way analysis of variance and Tukey's Multiple Comparison Test (p&lt;0.05). Results and Conclusion: Our results showed significant impairment in short-term recognition and spatial memory of group III and IV adolescent wistar rats when compared with the control (p&lt;0.05) and the formation of neurofibrillary tangle-like structures in neurons of the studied regions. .

Alzheimer-like cell death after vanadium pentoxide inhalation

Vanadium (V) toxicity depends on its oxidation state; it seems that vanadium pentoxide (V2O5) is the most toxic to the living cells. It has been reported that oral administration induces changes in motor activity and learning; in rats, I.P. administration increases lipid peroxidation levels in the cerebellum and the concentration of free radicals in the hippocampus and cerebellum. Mice that inhaled V2O5 presented a reduced number of tubulin+ in Leydig and Sertoli cells; it has also been reported that inhaled V2O5 induces loss of dendritic spines, necrosis, and hippocampus neuropil alterations; considering the direct consequence of the interaction of V with cytoskeletal components, makes us believe that V2O5 exposure could cause neuronal death in the hippocampus similar to that seen in Alzheimer disease. This work aimed to determine pyramidal hippocampal CA1 cytoskeletal alterations with Bielschowsky stain in rats exposed to V2O5. Male Wistar rats inhaled 0.02 M of V2O5 one h two times a week for two and six months. We found that rats, which inhaled V2O5 reached 56,57% of dead neurons after six months of inhalation; we recognize strong argyrophilic and collapsed somas and typical flame-shaped in all V-exposed rats hippocampus CA1 compared to controls. We also observe somatodendritic distortions. Axons and dendrites displayed thick dark bands replaced by noticeable thickening and nodosities and the cytoskeleton fibrillary proteins' linear traces. Our findings suggest that V2O5 inhalation induces Alzheimer-like cell death with evident cytoskeletal alterations.

Possible Preventive Effect of Donepezil and Hyoscyamoside by Reduction of Plaque Formation and Neuroinflammation in Alzheimer's Disease.

Background:Alzheimer disease (AD) is the most common age-dependent dementia. The complex natural accumulation of amyloid beta (Aβ) precursor protein in hippocampus neurons is regarded as the earliest pathological feature of AD, although there are cholinergic assumptions and effective inflammation in AD. In this animal experimental study, we evaluated the preventive effect of hyoscyamoside (Hyo) and donepezil (Dz) on plaque formation and improvement of neurogenic inflammation in AD rats.Methods:Dz was prepared and Hyo (steroidal saponin) was isolated from Hyoscymus niger. Then, Wistar rats divided into five groups including negative and positive controls, AD, Dz, and Hyo treatment groups based on the drug exposure and their behavioral alternation was examined using Morris water maze (MWM) test. Bielschowsky staining was used to detect the nerve fibers. Serum levels of interleukin (IL)-4 and IL-6 were evaluated by ELISA. The RNA expression of cyclin-dependent kinase CDK11-P58 in peripheral blood lymphocytes was performed using quantitative PCR.Results:The MWM test showed significant changes in time the models spent to find the hidden platform. The Hyo treatment group showed a notable speed change (P < 0.01). The histopathological analysis of the hippocampal tissue revealed the inhibition of Aβ formation in the treatment groups. The treatment groups had a significant decline in the serum level of IL-6, and the IL-4 serum level was increased in the Hyo and Dz treated groups. The expression levels of CDK11-P58 was significantly decreased in the treatment groups.Conclusions:In sum, the therapeutic effects of Hyo is comparable with that of Dz in AD rats by suppressing neuroinflammation. Thus, these compounds could be considered as a preventive agent in the AD therapy.

A New Model of Repetitive Traumatic Brain Injury in Mice.

Repetitive traumatic brain injury (rTBI) is a major health care concern that causes substantial neurological impairment. To better understand rTBI, we introduced a new model of rTBI in mice induced by sudden rotation in the coronal plane combined with lateral translation delivered twice at an interval of 24 h. By routine histology, histological examination of Prussian blue-stained sections revealed the presence of microbleed in the corpus callosum and brain stem. Amyloid precursor protein (β-APP) and neurofilament heavy-chain (NF-200) immunohistochemistry demonstrated axonal injury following rTBI. Swelling, waving, and enlargement axons were observed in the corpus callosum and brain stem 24 h after injury by Bielschowsky staining. Ultrastructural studies by electron microscopy provided further insights into the existence and progression of axonal injury. rTBI led to widespread astrogliosis and microgliosis in white matter, as well as significantly increased levels of tumor necrosis factor (TNF)-α and interleukin (IL)-1β. rTBI mice showed a significantly increased loss of righting reflex (LRR) duration within each time point compared with that of sham animals, which was under 15 min. rTBI mice exhibited depression-like behavior at 1 month. rTBI mice also demonstrated deficits in MWM testing. These results suggested that this model might be suitable for investigating rTBI pathophysiology and evaluating preclinical candidate therapeutics.

Use of a combination strategy to improve neuroprotection and neuroregeneration in a rat model of acute spinal cord injury.

Spinal cord injury is a very common pathological event that has devastating functional consequences in patients. In recent years, several research groups are trying to find an effective therapy that could be applied in clinical practice. In this study, we analyzed the combination of different strategies as a potential therapy for spinal cord injury. Immunization with neural derived peptides (INDP), inhibition of glial scar formation (dipyridyl: DPY), as well as the use of biocompatible matrix (fibrin glue: FG) impregnated with bone marrow mesenchymal stem cells (MSCs) were combined and then its beneficial effects were evaluated in the induction of neuroprotection and neuroregeneration after acute SCI. Sprague-Dawley female rats were subjected to a moderate spinal cord injury and then randomly allocated into five groups: 1) phosphate buffered saline; 2) DPY; 3) INDP + DPY; 4) DPY+ FG; 5) INDP + DPY + FG + MSCs. In all rats, intervention was performed 72 hours after spinal cord injury. Locomotor and sensibility recovery was assessed in all rats. At 60 days after treatment, histological examinations of the spinal cord (hematoxylin-eosin and Bielschowsky staining) were performed. Our results showed that the combination therapy (DPY+ INDP + FG + MSCs) was the best strategy to promote motor and sensibility recovery. In addition, significant increases in tissue preservation and axonal density were observed in the combination therapy group. Findings from this study suggest that the combination theapy (DPY+ INDP + FG + MSCs) exhibits potential effects on the protection and regeneration of neural tissue after acute spinal cord injury. All procedures were approved by the Animal Bioethics and Welfare Committee (approval No. 178544; CSNBTBIBAJ 090812960) on August 15, 2016.

Identification and immunophenotype of abnormal cells present in focal cortical dysplasia type IIb

BackgroundFocal cortical dysplasias (FCDs) are malformations of cortical development that present cortical dyslamination and abnormal cell morphology and are frequently associated with refractory epilepsy. FCD type IIb presents dysmorphic neurons (DNs) and balloon cells (BCs), which are the hallmarks of this dysplasia. Moreover, hypertrophic neurons (HyNs) may be present in FCD types I, II and III. The objective of this study was to perform a detailed morphology and immunophenotype study of BCs, DNs, and HyNs in a cohort of FCD IIb patients.MethodsCortices resected as a treatment for refractory epilepsy from 18 cases of FCD type IIb were analysed using Bielschowsky method and haematoxylin and eosin as routine stains. Immunophenotype was performed using specific antibodies to detect epitopes differentially expressed by abnormal cells.ResultsAll cases showed cortical dyslamination, BCs, DNs, and HyNs. No cell layer or column could be identified, except for cortical layer I. Lesions predominated in the frontal cortex (11 cases). DNs were large neurons and presented a clumped and or displaced Nissl substance towards the cell membrane, and a cytoplasm accumulation of neurofilament that displaced the nucleus to the cell periphery, as shown by Bielschowsky staining and immunohistochemistry. HyNs were as large as DNs, but without alterations of Nissl substance or dense neurofilament accumulation, with a central nucleus. BCs were identified as large, oval-shaped and pale eosinophilic cells, which lacked the Nissl substance, and presented an eccentric nucleus. BCs and DNs expressed epitopes of both undifferentiated and mature cells, detected using antibodies against nestin, vimentin, class III β-tubulin, pan-neuronal filaments, neurofilament proteins, β-tubulin and NeuN. Only BCs expressed GFAP.ConclusionFCDs present with disorganization of the cerebral cortex architecture, abnormal cell morphology, are frequently associated with refractory epilepsy, and their post-surgical prognosis depends on the type of FCD. The diagnosis of focal cortical dysplasia in a surgical specimen relies on the identification of the abnormal cells present in a dysplastic cortex specimen. The current report contributes to the identification of balloon cells, dysmorphic and hypertrophic neurons in the context of focal cortical dysplasia type IIb.

Quantitative assessment of demyelination in ischemic stroke in vivo using macromolecular proton fraction mapping

A recent MRI method, fast macromolecular proton fraction (MPF) mapping, was used to quantify demyelination in the transient middle cerebral artery occlusion (MCAO) rat stroke model. MPF and other quantitative MRI parameters (T1, T2, proton density, and apparent diffusion coefficient) were compared with histological and immunohistochemical markers of demyelination (Luxol Fast Blue stain, (LFB)), neuronal loss (NeuN immunofluorescence), axonal loss (Bielschowsky stain), and inflammation (Iba1 immunofluorescence) in three animal groups (n = 5 per group) on the 1st, 3rd, and 10th day after MCAO. MPF and LFB optical density (OD) were significantly reduced in the ischemic lesion on all days after MCAO relative to the symmetrical regions of the contralateral hemisphere. Percentage changes in MPF and LFB OD in the ischemic lesion relative to the contralateral hemisphere significantly differed on the first day only. Percentage changes in LFB OD and MPF were strongly correlated (R = 0.81, P < 0.001) and did not correlate with other MRI parameters. MPF also did not correlate with other histological variables. Addition of T2 into multivariate regression further improved agreement between MPF and LFB OD (R = 0.89, P < 0.001) due to correction of the edema effect. This study provides histological validation of MPF as an imaging biomarker of demyelination in ischemic stroke.

Correlation Between Neurological Deficits and Spinal Cord Pathological Changes in a Mouse Model of Multiple Sclerosis

Multiple sclerosis (MS) is a common neuroinflammatory disease causing a wide spectrum of clinical signs and symptoms. Neuropathological changes in MS including inflammation, demyelination and axonal degeneration are seen in the animal counterpart of MS, experimental autoimmune encephalomyelitis (EAE) and are biomarkers to follow the pathophysiology and any pharmacology of MS in experimental studies. To elucidate the pattern of these pathological abnormalities in EAE, different aspects of pathological findings and their correlations were studied in the model. EAE induction was done using myelin oligodendrocyte glycoprotein (MOG) 35–55 in C57BL/6 mice and hematoxylin and eosin, Luxol-fast-blue and Bielschowsky staining were used for histopathological evaluation in the lumbar, thoracic and cervical spinal cord. There were significant positive correlations between neurological disease score in EAE mice and each of these pathological findings: inflammation score, demyelination score and axonal degeneration. These correlations were observed regardless of the anatomic regions studied. Inflammation and demyelination scores were significantly associated. Degeneration and demyelination scores were correlated positively also. However, no statistically significant correlation was found between scores of inflammation and degeneration in neither of the three anatomical regions of the spinal cords of EAE mice. This study optimizes the EAE model regarding pathological findings in correlation with neurological deficits in the model. The results will help in better utility of the model in MS research.

188. AAV Mediated Gene Therapy to Modulate Neurotropic Factors in the Retina and in Neuronal Cells in Culture

Neurotrophic factors, particularly Brain Derived Neurotrophic Factor (BDNF) and its high affinity receptor TrkB, play an important role in protecting the retinal ganglion cells (RGCs) in glaucoma. Translation of the immense neuro-protective potential of BDNF/TrkB activation has been largely unsuccessful and gene therapy to modulate BDNF and TrkB have also shown only short-lived protective effects on the RGCs. PTPN11 phosphatase plays a key role in the regulation of BDNF/TrkB signaling in the retina. Here, we investigated the effects of PTPN11 modulation involving both over-expression and knock-down paradigms on the TrkB activation. PTPN11 and PTPN11-shRNA sequences were separately incorporated into the AAV2 vector under cytomegalovirus chicken β actin hybrid promoter linked with green fluorescent protein (GFP). SH-SY5Y cells were transfected with each of the PTPN11 modulating and control GFP constructs. PTPN11 over-expression resulted in significant reduced TrkB phosphorylation and accordingly reduced viability of the SH-SY5Y cells was observed while PTPN11 knockdown did not exhibit any significant effects on either TrkB phosphorylation or cellular survival. PTPN11 over-expression in neuronal cells was also accompanied by elevation of GADD, PERK and XBP-1 endoplasmic reticulum (ER) stress marker proteins as detected using western blotting. The roles of PTPN11 and TrkB in mediating ER stress were evaluated by treating cells with PTPN11 inhibitor (PHPS1) or TrkB agonist (7,8-dihydroxyflavone) independently, both of which ablated the up-regulation of ER stress proteins upon PTPN11 over-expression. The effects of PTPN11 over-expression on the rat retina were evaluated by administering PTPN11 AAV2 construct along with corresponding GFP controls individually into the rat eyes. Briefly, 5µl of AAV2 constructs were injected intravitreally into SD rats (2×1010 vg). Anti-GFP staining in retinal sections demonstrated AAV2 transduction in the RGC layer. Anti-NeuN/ anti-PTPN11 staining confirmed the ganglion cell specific over-expression of PTPN11. Rat retinas were assessed for inner retinal functional changes using scotopic threshold response (STR) measurements. Retinal structural changes were assessed using histological analysis. Results indicate that pSTR amplitude which primarily reflects the function of the RGCs was significantly diminished in the PTPN11 over-expression model. Hematoxylin and eosin staining also revealed degenerative changes primarily associated with the inner retina. Assessment of optic nerve sections using Bielschowsky's staining further identified reduced axonal density. Concluding, our findings strongly indicate that PTPN11 genetic modulation holds the promise to regulate neuronal cell survival through its effects on TrkB activation. PTPN11 over-expression exerts detrimental impact on the inner retinal health which has significant implications in glaucoma and other retinal disorders. Further studies involving rescue of the disease phenotype using PTPN11 knockdown in animal models will substantiate these observations and provide mechanistic insights into the roles of neurotrophic factor regulation in retina.

The role of CNS TLR2 activation in mediating innate versus adaptive neuroinflammation

Toll-like receptor 2 (TLR2) is expressed on immune cells in the periphery and the CNS and mediates both innate and adaptive immune responses. Recent studies have implicated TLR2 in systemic pathogenesis of adaptive immunity in experimental autoimmune encephalomyelitis (EAE). In addition, TLR2 is expressed on oligodendrocyte progenitor cells and its activation inhibits their differentiation and myelination. We investigated the roles of CNS TLR2 activation in mediating neuro-inflammatory responses in intact versus EAE animals. We examined the effects of intra-cerebro-ventricular (ICV) injection of Zymosan, a TLR2 agonist, on naive versus EAE animals. The neuro-inflammatory response was characterized by immune-fluorescent staining for IBA-1+ microglia/macrophages and CD3+ T cells, and by semi-quantitative real time PCR for TLR2 and immune cytokines. The nature of the immune cells isolated from EAE brain tissue was assessed by their proliferative response to the PLP peptide autoantigen. Survival and clinical scores were monitored; demyelination and axonal loss were quantified by Gold-Black and Bielschowsky stains. Our findings showed that Zymosan injection in naïve mice induced a massive neuro-inflammatory response without any clinical manifestations. In EAE mice, ICV Zymosan induced a severe acute toxic response with 80% mortality. Surviving animals returned to pre-injection clinical score, and their course of disease was not altered as compared to control EAE group. Demyelination and axonal loss were not affected by ICV Zymosan injection. Quantification of immune response in the brain by real time PCR, immunofluorescent stains and proliferative response to PLP peptide suggested that TLR2 activation induces innate but not adaptive immune response. We conclude that EAE mice are hypersensitive to CNS TLR2 activation with a severe toxic response. This might represent the susceptibility of multiple sclerosis patients to even trivial infections. As CNS TLR2 activation does not alter the clinical and pathological course of EAE, it implies that CNS TLR2 activation affects the innate but not adaptive brain immune responses.

Bone marrow stromal cell transplantation through tail vein injection promotes angiogenesis and vascular endothelial growth factor expression in cerebral infarct area in rats

Background aimsThis study sought to identify correlations between vascular endothelial growth factor (VEGF) expression after tail-vein injection of rat-derived bone marrow stromal cells (BMSCs) and neurogenesis and angiogenesis in cerebral infarct of rats. MethodsRats with intraluminal middle cerebral artery occlusion were injected in a tail vein with Hoechst-labeled BMSCs. Functional recovery from cerebral infarction was observed through the use of a locomotion score. The brains of injected rats were sliced, and Hoechst-labeled BMSCs in the infarct and peri-infarct areas and subventricular zone (SVZ) were detected with the use of fluorescence microscopy. Ki-67 (as a cell proliferation marker) and VEGF expression were determined by means of immunohistochemistry. Neurofibril formation and angiogenesis were examined by means of Bielschowsky staining. ResultsWithin 1 to 2 weeks after BMSC injection, rats showed significantly improved locomotion scores compared with rats without BMSC injection (P < 0.01). Viable BMSCs were found in the peri-infarct area. The numbers of Ki-67–positive and VEGF-positive cells in the peri-infarct area and SVZ of injected rats were significantly increased compared with the control group (P < 0.01). Numerous new vessels, neurofibrils and anastomosed vasculatures were present in the infarct area. These neurofibrils mainly surrounded the neovasculatures. ConclusionsThese results indicate that BMSC-transplantation in rats through tail vein injection can increase neurogenesis, perhaps as the result of VEGF-mediated and/or Ki-67–mediated angiogenesis.

A novel pathogenic mechanism for cerebellar lesions produced by Solanum bonariense in cattle.

Intoxication with Solanum bonariense in cattle causes cerebellar cortical degeneration with perikaryal vacuolation, axonal swelling, and death primarily of Purkinje cells, with accumulation of electron-dense residual storage bodies in membrane-bound vesicles. The pathogenesis of this disease is not fully understood. Previously, we proposed that inhibition of protein synthesis in Purkinje cells among other altered metabolic pathways could lead to cytoskeletal alterations, subsequently altering cell-specific axonal transport. In the present study, immunohistochemical and histochemical methods were used to identify neuronal cytoskeletal alterations and axonal loss, demyelination, and astrogliosis in the cerebellum of intoxicated bovines. Samples of cerebellum from 3 natural and 4 experimental cases and 2 control bovines were studied. Immunoreactivity against neurofilament (NF)-200KDa confirmed marked loss of Purkinje neurons, and phospho-NF protein, β-tubulin, and affinity reaction against phalloidin revealed an altered perikaryal distribution of neuronal cytoskeletal proteins in the remaining Purkinje cells in intoxicated cattle. Reactive astrogliosis in every layer of the cerebellar cortex was also observed with anti-glial fibrillary acidic protein immunohistochemistry. In affected cattle, demyelination and axonal loss in the cerebellar white matter, as well as basket cell loss were demonstrated with Klüver-Barrera and Bielschowsky stains, respectively. Based on these results, we propose that neuronal cytoskeletal alterations with subsequent interference of the axonal transport in Purkinje cells may play a relevant role in the pathogenesis of this neurodegenerative disorder, and also that demyelination and axonal loss in the cerebellar white matter, as well as astrogliosis in the gray matter, likely occur secondarily to Purkinje cell degeneration and death.

The experimental antiphospholipid syndrome: an invaluable tool to study autoimmunity-induced neurodegeneration

cells and its activation inhibits their differentiation and remyelination. These suggest a possible role of CNS TLR2 in progressive autoimmune demyelination. Methods: We examined the effects of intra-cerebro-ventricular (ICV) injection of Zymozan, a TLR2 agonist, on the clinical and pathological course of EAE. The survival and clinical scores were monitored; demyelination and axonal loss were quantified by gold-black and Bielschowsky stains, and the nature of neuro-inflammatory response was characterized by TLR2, IBA-1 and CD3 stainings and PCR for immune cytokines. Immune cells were isolated from EAE brain tissue and their proliferative response to the autoantigen (PLP peptide) or Concavallin A was examined in vitro. Results: Zymozan injection in naive mice induced a strong neuroinflammatory response without any clinical manifestations. In EAE mice, ICV Zymozan induced a severe acute toxic response with 80% mortality. Surviving animals returned to pre-injection clinical score, and their course of disease and CNS inflammatory parameters were not altered as compared to control EAE group. Demyelination and axonal loss were not affected by ICV Zymozan injections. Quantification of immune response in the brain by real time PCR, immunofluorescent stains and proliferative response to PLP peptide and ConA indicated an increase in innate but not adaptive immune response. Conclusions: (1) EAE mice are hypersensitive to CNS TLR2 activation with a severe toxic response. This might represent the susceptibility of multiple sclerosis patients to even trivial infections. (2) CNS TLR2 activation does not alter the clinical and pathological course of EAE. (3) These findings imply that CNS TLR2 activation affects the innate but not adaptive brain immune responses.

Behavioral and pathological changes in a transgenic mouse model of Alzheimer's disease

Alzheimer's disease (AD), a neurodegenerative brain disorder, is the most common cause of dementia, characterized by amyloid-β plaque accumulation, intracellular tangles and neuronal loss in selective brain regions. The frontal cortex, important for executive functioning, is one of the regions affected. In this communication, it has been investigated that learning and memory function and pathological changes of APPswe/PS1ΔE9 (APP/PS1) double transgenic mouse. Ten male APP/PS1 double transgenic mice and 10 male CS7BL/GJ mice were used in this study. The learning and memory functions were studied by Morris water maze, the pathological changes in brain tissue by modified Bielschowsky and Nissl's staining and the distribution of Aâ plaques in the APP/PS1 mouse brain by immunohistochemistry using avidin-biotinylated complex staining. We found that the APP/PS1 mice provided novel insights into the regional selective vulnerability of the frontal cortex to Alzheimer's disease and therefore these mice may prove as useful animal models for understanding the pathogenesis of Alzheimer's disease in people.

Factors Secreted by Endothelial Progenitor Cells Enhance Neurorepair Responses after Cerebral Ischemia in Mice

Cell therapy with endothelial progenitor cells (EPCs) has emerged as a promising strategy to regenerate the brain after stroke. Here, we aimed to investigate if treatment with EPCs or their secreted factors could potentiate angiogenesis and neurogenesis after permanent focal cerebral ischemia in a mouse model of ischemic stroke. BALB/C male mice were subjected to distal occlusion of the middle cerebral artery, and EPCs, cell-free conditioned media (CM) obtained from EPCs, or vehicle media were administered one day after ischemia. Magnetic resonance imaging (MRI) was performed at baseline to confirm that the lesions were similar between groups. Immunohistochemical and histological evaluation of the brain was performed to evaluate angio-neurogenesis and neurological outcome at two weeks. CM contained growth factors, such as VEGF, FGF-b and PDGF-bb. A significant increase in capillary density was noted in the peri-infarct areas of EPC- and CM-treated animals. Bielschowsky’s staining revealed a significant increase in axonal rewiring in EPC-treated animals compared with shams, but not in CM-treated mice, in close proximity with DCX-positive migrating neuroblasts. At the functional level, post-ischemia forelimb strength was significantly improved in animals receiving EPCs or CM, but not in those receiving vehicle media. In conclusion, we demonstrate for the first time that the administration of EPC-secreted factors could become a safe and effective cell-free option to be considered in future therapeutic strategies for stroke.