Mages except D. For D, of totally differentiated neurons and astrocytes was analyzed by immunostaining (D). The appearancethe scale bar is 200 m. with MAP2 (E) and GFAP (F), respectively. The scale bar is 100 (showed in panel (F)) for all of the three.three. CCI Induces Astrogliosis and Reduces Neurons in COs pictures except (D). For (D), the scale bar is 200 .To model TBI in COs, we delivered the influence into COs embedded inside the mouse skull and supported by the phantom brain. CCI was performed in COs at 220 DIV working with our newly adapted technique. As sham controls, we placed the COs within the skull filled with the phantom brain with out the impact. The CCI approach is well-established to model moderate to serious TBI in mouse. Hence, as a positive control, we also applied CCI into a live mouse brain to evaluate with COs. To assess astrogliosis, we performed immunofluorescence analysis using glial fibrillary acid protein (GFAP) as an astrocyte marker to evaluateCells 2021, 10, 2683 Cells 2021, ten, x FOR PEER REVIEW9 of 16 11 ofFigure 3. Astrogliosis and reduction of neurons in COs right after CCI. (A) Microphotographs of COs and mice brain subjected to Figure 3. Astrogliosis and reduction of neurons in COs after CCI. (A) Microphotographs of COs and mice brain subjected to CCI stained with GFAP and MAP2 (S)-(+)-Dimethindene Autophagy antibodies to recognize astrocytes and neurons, respectively. Immunostaining was CCI stained with GFAP and MAP2 antibodies to recognize astrocytes and neurons, respectively. Immunostaining was accomplished performed 7 days just after CCI. (B) Immunofluorescence quantifications of GFAP in mouse brain (Controls eight.241 two.five vs. CCI 96.68 7 days soon after CCI. (B) Immunofluorescence quantifications of GFAP in mouse brain (Controls eight.241 2.five vs. CCI 96.68 ten.7; ten.7; p = 0.0002) and (C) COs (Controls 67.31 five.0 vs. CCI 201.six 65; p = 0.0241). MAP2-positive neuronal density in (D) p = 0.0002) and (C) COs (Controls vs. CCI 26.24 12.five; p = 65; p = 0.0241). MAP2-positive neuronal density in (D) 7.0; mouse brain (Control 144.two 21.7 67.31 5.0 vs. CI 201.60.0012) and in COs (E) (Control 108.7 11.9 vs. CCI 40.73mouse brain (Iprodione Reactive Oxygen Species Handle 144.two 21.7 changes in astrocytes p COs and mouse brains have been observed 7 days immediately after CCI Magnificap = 0.001). (F) Morphological vs. CCI 26.24 12.5; of= 0.0012) and in COs (E) (Control 108.7 11.9 vs. CCI. 40.73 7.0; p = X40, scale bars = 50 m. adjustments in astrocytes of COs and mouse brains have been observed p 0.01; p 0.001. tion:0.001). (F) Morphological Statistical evaluation performed with Student’s t-test, p 0.05; 7 days right after CCI. Magnification: X40, scale bars = 50 . Statistical evaluation performed with Student’s t-test, p 0.05; p 0.01; p 0.001.3.4. Elevated Neuronal Harm in COs soon after CCICells 2021, ten,Cells 2021, ten, x FOR PEER Overview 12 of10 of3.4. Elevated Neuronal Harm in COs just after CCI Neuronal harm is one of hallmark primary pathological characteristics of TBI. We Neuronal harm is among the the hallmark main pathological attributes of TBI. We analyzed neuronal damage in COs, 7 days CCI CCI working with neuron-specific enolase analyzed neuronal damage in COs, 7 days afterafter making use of neuron-specific enolase (NSE). (NSE). NSE, an enzyme involved in glycolysis, has been reported as of late neural late NSE, an enzyme involved in glycolysis, has been reported as a marker a marker of mat- neural uration [41] and isand is regarded as a biomarker that will straight assess functionalto maturation [41] viewed as a biomarker that may directly assess functional harm damage neurons [42,.