Journal: bioRxiv

Article Title: NOD2 activation reprograms infiltrating inflammatory monocytes in the Zika virus infected CNS to maintain neural correlates of learning and memory

doi: 10.64898/2026.05.19.726300

Figure Lengend Snippet: (A) Representative images from ZIKV or mock-infected animals with either iMDP or MDP treatment, harvested at 10 dpi, and stained for detection of pre-synaptic (Synaptophysin, red) or post-synaptic (Homer1, green) termini in the CA3 region of the hippocampus. (B) Quantitation of Synaptophysin+, Homer1+, or co-localized puncta at 10 dpi. Mock iMDP n=3, Mock MDP n=4, ZIKV iMDP n=4, ZIKV MDP n=4. (C) Representative images from ZIKV or mock-infected animals with either iMDP or MDP treatment, harvested at 25 dpi, and stained for detection of pre-synaptic (Synaptophysin, red) or post-synaptic (Homer1, green) termini in the CA3 region of the hippocampus. (D) Quantitation of Synaptophysin+, Homer1+, or co-localized puncta at 25 dpi. Mock iMDP n=3, Mock MDP n=4, ZIKV iMDP n=6, ZIKV MDP n=6. (E) Representative images from ZIKV or mock-infected animals with either iMDP or MDP treatment, harvested at 25 dpi, and stained for detection of DCX (green), NeuN (grey), or BrdU (red), Location of insets are indicated in the image. DCX+/NeuN+/BrdU+ cells are indicated by arrows. (F) Quantification of NeuN+/BrdU+, and DCX+/NeuN+/BrdU+ cells within the dentate gyrus of the hippocampus at 25dpi. Mock iMDP n=4, Mock MDP n=4, ZIKV iMDP n=4, ZIKV MDP n=4. Scale bar, 25 µm (A, C), 100 µm (E). Each dot represents an individual mouse. Results are displayed as mean ± s.d. and were analyzed by two-way ANOVA, and corrected for multiple comparisons. All panels, * p <0.05, ** p <0.01, ***p< 0.001.

Article Snippet: To determine whether NOD2 activation impacts synapse engulfment by infiltrating macrophages or microglia, we quantified Homer1 puncta within Iba1+/TMEM119+ microglia versus Iba1+/TMEM119- macrophages within Lamp1+ lysosomes via Z-stack images from confocal microscopy with 3D mask rendering within the CA3 region using Imaris software( ).

Techniques: Infection, Staining, Quantitation Assay

Journal: bioRxiv

Article Title: NOD2 activation reprograms infiltrating inflammatory monocytes in the Zika virus infected CNS to maintain neural correlates of learning and memory

doi: 10.64898/2026.05.19.726300

Figure Lengend Snippet: (A-B) 8-week-old C57BL/6J animals were inoculated intracranially with ZIKV-Dakar (1x10 4 FFU) and administered an inactive isomer of MDP (iMDP, 10mg/kg) or MDP (10mg/kg) i.p. starting at day 7 post-infection and given daily injections until harvest (10 dpi). (A) Representative images from ZIKV or mock-infected animals with either iMDP or MDP treatment, harvested at 10 dpi, and stained for detection of pre-synaptic (Synaptophysin, red) or post-synaptic (Homer1, green) termini in the CA3 region of the hippocampus. (B) Quantitation of Synaptophysin+, Homer1+, or co-localized puncta at 10 dpi. Scale bar, 25 µm. Each dot represents an individual mouse; n=5 per group. Results are displayed as mean ± s.d. of fold change of mock, iMDP-treated animals. Raw puncta values were used for statistical analysis and analyzed by two-way ANOVA, corrected for multiple comparisons.

Article Snippet: To determine whether NOD2 activation impacts synapse engulfment by infiltrating macrophages or microglia, we quantified Homer1 puncta within Iba1+/TMEM119+ microglia versus Iba1+/TMEM119- macrophages within Lamp1+ lysosomes via Z-stack images from confocal microscopy with 3D mask rendering within the CA3 region using Imaris software( ).

Techniques: Infection, Staining, Quantitation Assay

Journal: bioRxiv

Article Title: NOD2 activation reprograms infiltrating inflammatory monocytes in the Zika virus infected CNS to maintain neural correlates of learning and memory

doi: 10.64898/2026.05.19.726300

Figure Lengend Snippet: (A) Representative immunostaining of hippocampal CA3 and (B) quantification of colocalized presynaptic and postsynaptic puncta using synaptophysin (red) and Homer-1 (green), respectively, at 10 dpi in generated bone marrow chimeric mice (WT donor – WT recipient (iMDP n =5, MDP n =5), Nod2 KO donor – WT recipient (iMDP n =5, MDP n =5), WT donor – Nod2 KO recipient (iMDP n =5, MDP n =6), Nod2 KO donor - Nod2 KO recipient (iMDP n =3, MDP n =4). (C) Representative imaging of hippocampal CA3 region for Homer1 (magenta), Iba1 (cyan), Lamp1 (yellow), and TMEM119 (grey). Imaris 3D rendering of the same image is shown with magnified insets labeled. Arrows indicate colocalized Homer1+ puncta within Lamp1+/Iba1+/TMEM119+ masks. (D, E) Quantitative analysis of macrophage (TMEM119-/Iba1+; D) and microglial (TMEM119+/Iba1+; E) volume, Lamp1+ volume within each cell type, and colocalized Homer1+ puncta with Lamp1+ masks within each cell type at 10 dpi. Data were pooled from two independent experiments. Scale bars, 25 µm (A, C; unless otherwise indicated in Imaris rendering). Data are displayed as mean ± s.d. and were analyzed by students’s t-test (B, D, E). All panels, ** p <0.01, ***p< 0.001, **** p < 0.0001.

Article Snippet: To determine whether NOD2 activation impacts synapse engulfment by infiltrating macrophages or microglia, we quantified Homer1 puncta within Iba1+/TMEM119+ microglia versus Iba1+/TMEM119- macrophages within Lamp1+ lysosomes via Z-stack images from confocal microscopy with 3D mask rendering within the CA3 region using Imaris software( ).

Techniques: Immunostaining, Generated, Imaging, Labeling

Journal: Brain, Behavior, & Immunity - Health

Article Title: Microglial response to sleep deprivation depends on the hippocampal region and paradigm used in adult male mice

doi: 10.1016/j.bbih.2026.101213

Figure Lengend Snippet: Peripheral immune cell infiltration was not altered by acute SD. Epifluorescence images at 20x magnification displaying Iba1 (in red) and TMEM119 (in green) stained microglia in the CA1 SR (A, C, E) and CA3 SR (B, D, F) regions of the SD and control groups (A-F). Scale bar is equivalent to 100 μm. Acute SD had no detectable effect on peripheral immune cell infiltration in the CA1 and CA3 SR regions in male mice (G). White arrows: Iba1+/TMEM119-cells. Data are expressed as the mean ± standard error of the mean. n = 5 mice/group. Statistical analysis was performed with a Kruskal-Wallis test. CA: Cornu Ammonis, Iba1: ionized calcium-binding adaptor molecule 1, TMEM119: transmembrane protein 119, SD: sleep deprivation, SR: stratum radiatum . (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: For the density analysis of Iba1+ cells, between 4 and 5 mosaics per animal displaying the CA1 and CA3 SR regions were captured with a Basler ace acA1920-40uc camera (2.3 MP) and the Microvisoneer manual WSI software on a brightfield microscope using a 20x objective.

Techniques: Staining, Control, Binding Assay

Journal: Brain, Behavior, & Immunity - Health

Article Title: Microglial response to sleep deprivation depends on the hippocampal region and paradigm used in adult male mice

doi: 10.1016/j.bbih.2026.101213

Figure Lengend Snippet: Microglial density and distribution were modified differently based on the SD paradigm and examined hippocampal region. Representative brightfield pictures at 20x magnification showing Iba1-stained microglia in hippocampal CA1 SR (A, C, E) and CA3 SR (B, D, F) of control and SD groups. Scale bar is equivalent to 100 μm. Microglial density decreased for both SD paradigms in CA1 SR , but not in CA3 SR (G). Accordingly, NND was also increased in the CA1 SR for both conditions, compared to controls (Fig. H). Spacing index was unchanged in CA1 SR , but increased in CA3 SR by novelty exposure (I). Data are expressed as mean ± standard error of the mean. n = 5 mice/group. Statistical analysis was performed with one-way ANOVA, where ∗ p < 0.05,∗∗ p < 0.01,∗∗∗ p < 0.001,∗∗∗∗p < 0.0001. a. u.: arbitrary units, CA: Cornu Ammonis, NND: nearest-neighbour distance, Iba1: ionized calcium-binding adaptor molecule 1, SD: sleep deprivation, SR: stratum radiatum .

Article Snippet: For the density analysis of Iba1+ cells, between 4 and 5 mosaics per animal displaying the CA1 and CA3 SR regions were captured with a Basler ace acA1920-40uc camera (2.3 MP) and the Microvisoneer manual WSI software on a brightfield microscope using a 20x objective.

Techniques: Modification, Staining, Control, Binding Assay

Journal: Brain, Behavior, & Immunity - Health

Article Title: Microglial response to sleep deprivation depends on the hippocampal region and paradigm used in adult male mice

doi: 10.1016/j.bbih.2026.101213

Figure Lengend Snippet: Microglial morphology changed differently depending on the SD paradigm and hippocampal region. Representative brightfield pictures at 40x magnification showing Iba1+ stained microglia (tracing in red) in the hippocampal CA1 SR (A, C, E) and CA3 SR (B, D, F) of control and SD groups. Scale bar is equivalent to 12 μm. Microglial soma area (G) and perimeter (H) were decreased in the CA1 SR and the CA3 SR following SD by novelty exposure. Microglial arborization circularity (I) and solidity (J) were reduced after SD by novelty exposure, only in CA1 SR . Microglial arborization perimeter increased after SD by novelty exposure, also only in the CA1 (K). Morphological index was reduced with SD by novelty exposure in both CA1 and CA3 SR (L). Data are expressed as mean ± standard error of the mean. n = 5 mice/group. One-way ANOVA was performed as statistical analysis, with ∗ p < 0.05,∗∗ p < 0.01,∗∗∗ p < 0.001,∗∗∗∗p < 0.0001. CA: Cornu Ammonis, Iba1: ionized calcium-binding adaptor molecule 1, SD: sleep deprivation, SR: stratum radiatum . (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: For the density analysis of Iba1+ cells, between 4 and 5 mosaics per animal displaying the CA1 and CA3 SR regions were captured with a Basler ace acA1920-40uc camera (2.3 MP) and the Microvisoneer manual WSI software on a brightfield microscope using a 20x objective.

Techniques: Staining, Control, Binding Assay

Journal: Brain, Behavior, & Immunity - Health

Article Title: Microglial response to sleep deprivation depends on the hippocampal region and paradigm used in adult male mice

doi: 10.1016/j.bbih.2026.101213

Figure Lengend Snippet: Microglial phagolysosomal soma activity increased after SD by novelty exposure in CA1 SR . Representative confocal pictures at a 63x magnification showing microglia stained for Iba1 (in green- and CD68 (in red) in the hippocampal CA1 SR (A, C, E) and CA3 SR (B, D, F) of the control and SD groups. Scale bar is equivalent to 15 μm. CD68 puncta co-labelled with Iba1+ soma were increased in hippocampal CA1 SR in mice exposed to novelty exposure compared to control and SD by gentle handling (G). Data are expressed as mean ± standard error of the mean. n = 5 mice/group. One-way ANOVA where ∗∗ p < 0.01. CA: Cornu Ammonis, CD68: cluster of differentiation 68, Iba1: ionized calcium-binding adaptor molecule 1, SD: sleep deprivation, SR: stratum radiatum. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: For the density analysis of Iba1+ cells, between 4 and 5 mosaics per animal displaying the CA1 and CA3 SR regions were captured with a Basler ace acA1920-40uc camera (2.3 MP) and the Microvisoneer manual WSI software on a brightfield microscope using a 20x objective.

Techniques: Activity Assay, Staining, Control, Gentle, Binding Assay

Journal: Brain, Behavior, & Immunity - Health

Article Title: Microglial response to sleep deprivation depends on the hippocampal region and paradigm used in adult male mice

doi: 10.1016/j.bbih.2026.101213

Figure Lengend Snippet: Microglial co-labelling with the pre-synaptic marker VGLUT1 in the CA1 SR or CA3 SR was not modified after SD paradigms. Representative confocal microscopy images at 63x magnification showing Iba1+ microglia (green) with VGLUT1+ pre-synaptic puncta (red) in the CA1 SR (A, C, E) and CA3 SR (B, D, F) of the control and SD groups. Scale bar is equivalent to 10 μm. Both SD by gentle handling and novelty exposure did not alter the number of pre-synaptic puncta in the soma in the CA1 SR and CA3 SR (G) regions in mice. Data are expressed as mean ± standard error of the mean. One-way ANOVA followed by Tukey post-hoc tests were performed as statistical analysis. n = 5 mice/group, 13-19 Iba1+ cells/region/animal. CA = Cornu Ammonis, Iba1 = ionized calcium-binding adaptor molecule 1, SD: sleep deprivation, SR: stratum radiatum , VGLUT1: vesicular glutamate transporter. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: For the density analysis of Iba1+ cells, between 4 and 5 mosaics per animal displaying the CA1 and CA3 SR regions were captured with a Basler ace acA1920-40uc camera (2.3 MP) and the Microvisoneer manual WSI software on a brightfield microscope using a 20x objective.

Techniques: Marker, Modification, Confocal Microscopy, Control, Gentle, Binding Assay

Journal: Brain, Behavior, & Immunity - Health

Article Title: Microglial response to sleep deprivation depends on the hippocampal region and paradigm used in adult male mice

doi: 10.1016/j.bbih.2026.101213

Figure Lengend Snippet: Microglial ultrastructure differed between SD paradigms in the CA1 SR . Representative scanning electron microscopy images at 5 nm resolution showing examples of microglial cell bodies in the CA1 SR (A, C, E) and CA3 SR (B, D, F). Inset (E) shows a close-up of a fully digested phagosome. The scale bar is equivalent to 2 μm. Microglial cytoplasm circularity (G) and solidity (H) decreased following SD by novelty exposure compared to gentle handling in the CA1 SR . The number of fully digested phagosomes also increased in CA1 SR after novelty exposure compared to gentle handling. Quantitative analyses were performed at the level of individual microglial cells, using a nested design to account for within-animal dependency. Data are expressed as mean ± standard error of the mean. n = 10-11 microglia/mouse, N = 4 mice/group. Nested one-way ANOVA followed by Tukey post-hoc tests were performed, where ∗ p < 0.05 and ∗∗ p < 0.01. CA = Cornu Ammonis, SD: sleep deprivation, SR : stratum radiatum . red outline: cytoplasm membrane, yellow outline: nucleus, blue pseudocoloring: fully digested phagosome. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: For the density analysis of Iba1+ cells, between 4 and 5 mosaics per animal displaying the CA1 and CA3 SR regions were captured with a Basler ace acA1920-40uc camera (2.3 MP) and the Microvisoneer manual WSI software on a brightfield microscope using a 20x objective.

Techniques: Electron Microscopy, Gentle, Membrane