Impairment in the Intestinal Morphology and in the Immunopositivity of Toll-like Receptor-4 and Other Proteins in an Autistic Mouse Model

Abstract

Autism spectrum disorder (ASD) identifies a neurodevelopmental disease defined by social impairments and repetitive or stereotyped behaviors. The etiology of ASD remains unclear; it primarily affects the brain, but a link between gastrointestinal (GI) diseases, inflammatory mucosal pathology and this disorder has been suggested. In particular, a central role seems to be played by an imbalance in pro-and anti-inflammatory cytokines, oxidative stress, and apoptosis. Toll-like receptor 4 (TLR4) is a protein of innate immunity responsible for the regulation and maintenance of intestinal homeostasis. Through histochemical and immunohistochemical evaluations we analyzed the intestinal morphology and the immunopositivity of TLR4 and of other pro-inflammatory and apoptotic proteins in BTBR T+Itpr3tf/J mice. Morphological data showed that the mucosal tunica presented longer intestinal villi. The length of the villi and the epithelial surface determine the exchanges of the intestinal mucosa with luminal contents, modifying the microbiota composition. The biochemical and immunohistochemical results indicated a close relationship among the increase of TLR4 and the activation of NF-kB subunits (p65 and p50) and pro-inflammatory and apoptotic proteins, such as cyclooxygenase-2, interleukin-1β, inducible nitric oxide synthase, tumor nuclear factor—alpha, caspase-3, caspase-8. These preliminary results require more in-depth study but they suggest the TLR4 signaling pathway as a possible target for therapeutic approaches to reduce GI disorders in ASD.

Exposure to Methylmercury at Juvenile Stage Worsens Autism-like Symptoms in Adult BTBR T+tf/J Mice Due to Lack of Nuclear Factor Erythroid 2-Related Factor 2 Signaling Upregulation in Periphery and Brain

“…Over the past years, the association between oxidative stress and ASD has been intensively studied. For instance, BTBR mice and ASD subjects show upregulation of NF-kB, iNOS, NADPH oxidase, lipid peroxides, and nitrotyrosine which may exacerbate the autism-like symptoms by increasing oxidative damage in the periphery and brain [ 18 , 19 , 27 , 44 , 45 , 46 , 47 , 48 , 49 ]. Past studies showed that BTBR mice and individuals with ASD had increased lipid peroxidation and other markers of oxidative stress such as nitrotyrosine as compared to normal controls.…”

“…Past studies showed that BTBR mice and individuals with ASD had increased lipid peroxidation and other markers of oxidative stress such as nitrotyrosine as compared to normal controls. These oxidative events may exacerbate the autism-like symptoms in mice and human ASD subjects by increasing the oxidative damage in the periphery and brain [ 43 , 44 , 45 ]. A recent study showed that supplementation of a ketogenic diet alleviated oxidative stress by reducing the level of lipid peroxidation in the brain tissue of BTBR mice which was associated with improvements of autism-like behavior [ 38 ].…”

Relation Between Reactive Oxygen Species Production and Transient Receptor Potential Vanilloid1 Expression in Human Skin During Aging

“…To demonstrate the specificity of antibodies, we used a preadbsorption test: 1% bovine serum albumin and 0.05% Tween 20 for 1 hour at room temperature. 52 Skin sections were incubated for 45 minutes at 37°C and for 1 hour at room temperature with the following primary antibodies: rabbit polyclonal anti-TRPV1 (working concentration 1 µg/ml; diluted 1:350; Abcam, Cambrige, UK), 53 rabbit polyclonal anti-MMP9 (working concentration 1 µg/ml; diluted 1:400; Abbiotec, San Diego, CA, USA), rabbit polyclonal anticyclooxygenase-2 (COX-2) (working concentration 0.5 µg/ml; diluted 1:400; Cayman Chemical, Ann Arbor, MI, USA), 54 and mouse monoclonal antisuperoxide dismutase-1(SOD-1) (working concentration 2 µg/ml; diluted 1:100; Santa Cruz Biotechnology, Dallas, TX, USA). 55–57 Then, the sections were incubated for 1 hour with specific biotinylated secondary antibodies (Vector Laboratories, Newark, CA, USA) and subsequently conjugated with avidin-biotin peroxidase complex (Vector Laboratories, Burlingame, CA, USA).…”

Hepatic Alterations in a BTBR T + Itpr3tf/J Mouse Model of Autism and Improvement Using Melatonin via Mitigation Oxidative Stress, Inflammation and Ferroptosis

“…Then, the sections were washed in Tris-buffered saline (TBS) for 5 min and incubated in 3% hydrogen peroxide for 10 min at room temperature. To demonstrate the specificity of antibodies, we used a pre-absorption test (blocking agent): 1% bovine serum albumin in 0.05% Tween 20 for 1 h at room temperature [63]. Subsequently, liver sections were incubated for 1 h and 30 min at room temperature with the following primary antibodies: mouse monoclonal anti-SOD-1 (diluted 1:100; Santa Cruz Biotechnology, Dallas, TX, USA), rabbit polyclonal anti-CAT (diluted 1:200; Abcam, Cambridge, UK), mouse monoclonal anti-IL-1β (diluted 1:100; Santa Cruz Biotechnology, Dallas, TX, USA), mouse monoclonal anti-HO-1 (diluted 1:100; Santa Cruz Biotechnology, Dallas, TX, USA), mouse monoclonal anti-GPX4 (diluted 1:1000; Proteintech, Manchester, UK), rabbit monoclonal anti-NRF2 (diluted 1300; Proteintech, Manchester, UK) and rabbit monoclonal anti-KEAP1 (diluted 1:1000; Proteintech, Manchester, UK).…”