Improved immunocytochemical identification of neural, endothelial, and inflammatory cell types in paraffin-embedded injured adult rat spinal cord

Casella, Gizelda; Bunge, Mary Bartlett; Wood, Patrick M.

doi:10.1016/j.jneumeth.2004.04.008

Cited by 36 publications

(30 citation statements)

References 12 publications

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“…Coronal brain sections (40 m) were obtained with a cryostat (Microm HM 505E) and collected on polysine glass slides (Menzel-Gläser, Thermo Scientific). Brain sections were treated with citrate buffer, pH 6.0, for 15 min for epitope retrieval (Jiao et al, 1999;Shi et al, 2001;Casella et al, 2004) and incubated with a peroxide quenching solution and 3% methanol-hydroperoxide for 10 min, followed by a blocking solution (5% serum in 0.1% PBS/Tween 20) for 1 h. Sections were subsequently incubated overnight at 4°C with the following antibodies: rabbit polyclonal anti-GFAP (1:100; SigmaAldrich), rat monoclonal anti-CD11b (1:350; Abcam), and rabbit polyclonal anti-activated caspase-3 (1:100; Cell Signaling). Sections were then incubated for 30 min with the respective secondary biotinylated antibodies, against rabbit (1:1000; Sigma-Aldrich) or rat (1: 200; Vector Laboratories), respectively.…”

Section: Methodsmentioning

confidence: 99%

Pivotal Role of TLR4 Receptors in Alcohol-Induced Neuroinflammation and Brain Damage

Alfonso-Loeches

Pascual-Lucas²,

Blanco³

et al. 2010

Journal of Neuroscience

508

499

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Toll-like receptors play an important role in the innate immune response, although emerging evidence indicates their role in brain injury and neurodegeneration. Alcohol abuse induces brain damage and can sometimes lead to neurodegeneration. We recently found that ethanol can promote TLR4 signaling in glial cells by triggering the induction of inflammatory mediators and causing cell death, suggesting that the TLR4 response could be an important mechanism of ethanol-induced neuroinflammation. This study aims to establish the potential role of TLR4 in both ethanol-induced glial activation and brain damage. Here we report that TLR4 is critical for ethanol-induced inflammatory signaling in glial cells since the knockdown of TLR4, by using both small interfering RNA or cells from TLR4-deficient mice, abolished the activation of microtubule-associated protein kinase and nuclear factor-B pathways and the production of inflammatory mediators by astrocytes. Our results demonstrate, for the first time, that whereas chronic ethanol intake upregulates the immunoreactive levels of CD11b (microglial marker) and glial fibrillary acidic protein (astrocyte marker), and also increases caspase-3 activity and inducible nitric oxide synthase, COX-2, and cytokine levels [interleukin (IL)-1␤, tumor necrosis factor-␣, IL-6] in the cerebral cortex of female wild-type mice, TLR4 deficiency protects against ethanol-induced glial activation, induction of inflammatory mediators, and apoptosis. Our findings support the critical role of the TLR4 response in the neuroinflammation, brain injury, and possibly in the neurodegeneration induced by chronic ethanol intake.

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Section: Methodsmentioning

confidence: 99%

Pivotal Role of TLR4 Receptors in Alcohol-Induced Neuroinflammation and Brain Damage

Alfonso-Loeches

Pascual-Lucas²,

Blanco³

et al. 2010

Journal of Neuroscience

508

499

View full text Add to dashboard Cite

show abstract

“…Two coronal sections (20 μm thick) per animal were mounted on glass slides (TruBond 380, Tru Scientific, Bellingham, WA, USA) coated with an adhesive protein solution (Cell-Tak, Corning, NY, USA), and subjected to heat-induced antigen retrieval in pH 6.1 citrate buffer (DAKO, Carpinteria, CA, USA). Autofluorescence from aldehyde fixation and hemoglobin was reduced by sequential treatment with 0.1% sodium borohydride in PBS for 30 min and 0.1% Sudan black in 70% ethanol for 20 min [37]. Sections were blocked with 10% normal goat serum with 0.1% Triton-X, 5% BSA in PBS, incubated overnight with primary mouse monoclonal anti-3-NT antibody (1:100, #05-233, Millipore) followed by application of secondary goat anti-mouse IgG-conjugated Alexa Fluor 555 antibody (Life technologies).…”

Section: Methodsmentioning

confidence: 99%

Effects of Early Post-Ischemic Reperfusion and tPA on Cerebrovascular Function and Nitrosative Stress in Female Rats

Ahnstedt

Sweet

Cruden

et al. 2016

Transl. Stroke Res.

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Stroke is a major health issue in women. Our previous studies in male rats showed decreased myogenic tone in middle cerebral arteries (MCAs) after ischemia and reperfusion (I/R), while tone in parenchymal arterioles (PAs) was increased. This vascular response may aggravate stroke damage in males by limiting reperfusion, however, the effect in females is not known. The current study investigated the effect of I/R and tissue plasminogen activator (tPA) on myogenic tone and reactivity of MCAs and PAs in female rats. Nitrosative stress by peroxynitrite and recruitment of inflammatory neutrophils to the microvasculature were also studied. Female rats were subjected to 2 h MCA filament occlusion (n=16) or sham surgery (n=17) and given tPA (1 mg/kg, i.v) or vehicle followed by 30 min reperfusion. Myogenic tone and reactivity were measured in isolated and pressurized MCAs and PAs from the same animals. Cerebrovascular F-actin, 3-nitrotyrosine (3-NT, peroxynitrite marker), and intravascular neutrophils were quantified. Myogenic tone and constriction to the nitric oxide synthase inhibitor Nω-Nitro-L-arginine were decreased in MCAs but unchanged in PAs after I/R with no effect of tPA. F-actin and 3-NT expression were unaffected by I/R or tPA. Our study showed that MCAs from females, similar to what have been seen in males, are dilated after I/R and have decreased myogenic tone while tone in PAs was unchanged. Increased small vessel resistance may contribute to decreased reperfusion and worse outcome after stroke.

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“…The following antibodies were used: 1) anti-glial fibrillary acidic protein (GFAP) (1:500, rabbit polyclonal, DakoCytomation, Glostrup, Denmark) a marker for reactive astrogliosis, found elevated in human autism neuropathology studies [66] and in cerebrospinal fluid from autism patients [67] , 2) anti-rat CD68 antigen (1: 200, monoclonal, Serotec, Oxford, UK), 3) anti-IbA1 ( 1:10, 000, rabbit polyclonal, Wako, Richmond, VA) both markers for activated microglia and found elevated in human autism brain [66] and epilepsy [68] , 4) anti-cleaved caspase 3 (1: 100, rabbit polyclonal, Cell Signaling Technology, Danvers, MA), a marker for apoptotic cytotoxicity [69,70] and; 5) antiNeuN (1:1000, monoclonal, Chemicon USA ), a marker for neurons [71] . Tissue sections were mounted on glass slides (SurgiPath, Canada) and dried overnight at 37°C.…”

Section: Assessments Of Neuropathologymentioning

confidence: 99%

A Novel Rodent Model of Autism: Intraventricular Infusions of Propionic Acid Increase Locomotor Activity and Induce Neuroinflammation and Oxidative Stress in Discrete Regions of Adult Rat Brain

MacFabe¹,

Rodríguez-Capote²,

Hoffman³

et al. 2008

American J. of Biochemistry and Biotechnology

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Innate neuroinflammatory changes, increased oxidative stress and disorders of glutathione metabolism may be involved in the pathophysiology of autism spectrum disorders (ASD). Propionic acid (PPA) is a dietary and gut bacterial short chain fatty acid which can produce brain and behavioral changes reminiscent of ASD following intraventricular infusion in rats. Adult Long-Evans rats were given intraventricular infusions of either PPA (.26M, 4µl animal −1 ) or phosphate buffered saline (PBS)vehicle, twice daily for 7 days. Immediately following the second daily infusion, the locomotor activity of each rat was assessed in an automated open field (Versamax) for 30 min. PPA-treated rats showed significant increases in locomotor activity compared to PBS vehicle controls. Following the last treatment day, specific brain regions were assessed for neuroinflammatory or oxidative stress markers. Immunohistochemical analyses revealed reactive astrogliosis (GFAP), activated microglia (CD68, Iba1) without apoptotic cell loss (Caspase 3' and NeuN) in hippocampus and white matter (external capsule) of PPA treated rats. Biomarkers of protein and lipid peroxidation, total glutathione (GSH) as well as the activity of the antioxidant enzymes superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx), glutathione reductase (GR) and glutathione S-transferase (GST) were examined in brain homogenates. Some brain regions of PPA treated animals (neocortex, hippocampus, thalamus, striatum) showed increased lipid and protein oxidation accompanied by decreased total GSH in neocortex. Catalase activity was decreased in most brain regions of PPA treated animals suggestive of reduced antioxidant enzymatic activity. GPx and GR activity was relatively unaffected by PPA treatment while GST was increased, perhaps indicating involvement of GSH in the removal of PPA or related catabolites. Impairments in GSH and catalase levels may render CNS cells more susceptible to oxidative stress from a variety of toxic insults. Overall, these findings are consistent with those found in ASD patients and further support intraventricular PPA administration as an animal model of ASD.

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Improved immunocytochemical identification of neural, endothelial, and inflammatory cell types in paraffin-embedded injured adult rat spinal cord

Cited by 36 publications

References 12 publications

Pivotal Role of TLR4 Receptors in Alcohol-Induced Neuroinflammation and Brain Damage

Pivotal Role of TLR4 Receptors in Alcohol-Induced Neuroinflammation and Brain Damage

Effects of Early Post-Ischemic Reperfusion and tPA on Cerebrovascular Function and Nitrosative Stress in Female Rats

A Novel Rodent Model of Autism: Intraventricular Infusions of Propionic Acid Increase Locomotor Activity and Induce Neuroinflammation and Oxidative Stress in Discrete Regions of Adult Rat Brain

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