Pentraxin 3 contributes to neurogenesis after traumatic brain injury in mice

Abstract

Emerging evidence indicates that pentraxin 3 is an acute-phase protein that is linked with the immune response to inflammation. It is also a newly discovered marker of anti-inflammatory A2 reactive astrocytes, and potentially has multiple protective effects in stroke; however, its role in the adult brain after traumatic brain injury is unknown. In the present study, a moderate model of traumatic brain injury in mice was established using controlled cortical impact. The models were intraventricularly injected with recombinant pentraxin 3 (the recombinant pentraxin 3 group) or an equal volume of vehicle (the control group). The sham-operated mice underwent craniotomy, but did not undergo the controlled cortical impact. The potential neuroprotective and neuroregenerative roles of pentraxin 3 were investigated on days 14 and 21 after traumatic brain injury. Western blot assay showed that the expression of endogenous pentraxin 3 was increased after traumatic brain injury in mice. Furthermore, the neurological severity test and wire grip test revealed that recombinant pentraxin 3 treatment reduced the neurological severity score and increased the wire grip score, suggesting an improved recovery of sensory-motor functions. The Morris water maze results demonstrated that recombinant pentraxin 3 treatment reduced the latency to the platform, increased the time spent in the correct quadrant, and increased the number of times traveled across the platform, thus suggesting an improved recovery of cognitive function. In addition, to investigate the effects of pentraxin 3 on astrocytes, specific markers of A2 astrocytes were detected in primary astrocyte cultures in vitro using western blot assay. The results demonstrated that pentraxin 3 administration activates A2 astrocytes. To explore the protective mechanisms of pentraxin 3, immunofluorescence staining was used. Intraventricular injection of recombinant pentraxin 3 increased neuronal maintenance in the peri-injured cortex and ipsilateral hippocampus, increased the number of doublecortin-positive neural progenitor cells in the subventricular and subgranular zones, and increased the number of bromodeoxyuridine (proliferation) and neuronal nuclear antigen (mature neuron) double-labeled cells in the hippocampus and peri-injured cortex. Pentraxin 3 administration also increased the number of neurospheres and the number of bromodeoxyuridine and doublecortin double-labeled cells in neurospheres, and enhanced the proliferation of neural progenitor cells in primary neural progenitor cell cultures in vitro. In conclusion, recombinant pentraxin 3 administration activated A2 astrocytes, and consequently improved the recovery of neural function by increasing neuronal survival and enhancing neurogenesis. All experiments were approved by the Animal Ethics Committee of the First Affiliated Hospital of Chongqing Medical University, China on March 1, 2016.

Keywords: brain injury; brain trauma; cells; neurogenesis; plasticity; protein; recovery; regeneration.

Figure 1

Experimental timeline and PTX3 expression in the adult mouse brain after TBI. (A) Graphic experimental timeline of the in vivo studies. (B) Western blot image of PTX3 protein levels in the ipsilateral cortex at 6 hours and 1, 3, 7, 14, and 21 days following CCI; samples from the sham-operated group were used as controls. PTX3 expression in the perilesional cortex was upregulated at 6 hours; this increase lasted for 3 days and then the expression returned to baseline levels. GAPDH was used as the loading control. (C) Representative images of immunostaining for PTX3 on day 1 after CCI. PTX3 (green) was localized to Iba1-positive microglia (red, left image), GFAP-positive astrocytes (red, middle image), and neuronal nuclei antigen (NeuN)-positive neurons (red, right image) in the perilesional cortex. Cell nuclei were stained with DAPI (blue). However, there were no PTX3-positive cells in the sham-operated mice. n = 6 per group, scale bar: 40 μm. BrdU: Bromodeoxyuridine; CCI: controlled cortical impact; DAPI: 4',6-diamidino- 2-phenylindole; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFAP: glial fibrillary acidic protein; Iba1: ionized calcium-binding adaptor molecule 1; MWM: Morris water maze; NSS: neurological severity score; PTX3: pentraxin 3; rPTX3: recombinant pentraxin 3; TBI: traumatic brain injury.

Figure 2

rPTX3 activates A2 astrocytes. Western blot assay of the A2 reactive astrocyte markers PTX3, arginase-1, and Nrf2 after 100 ng/mL rPTX3 administration for 24 hours in primary astrocytes in vitro. GAPDH was used as an internal loading control. Representative images of PTX3, arginase-1, and Nrf2 protein expression (A) and the quantification data (B–D) reveal that these markers were significantly upregulated after rPTX3 treatment. ***P < 0.001. Data are expressed as the mean ± SEM (n = 6; Student's t-test). GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; Nrf2: nuclear factor-E2-related factor 2; PTX3: pentraxin 3; rPTX3: recombinant pentraxin 3.

Figure 3

rPTX3 increases neuronal maintenance after traumatic brain injury. Neuronal survival in the ipsilateral hippocampus and cortex was measured by immunostaining on day 14 after controlled cortical impact. (A) Neurons (neuronal nuclear antigen, NeuN; green) in the hippocampus (left, upper) and an enlarged view of the hippocampal CA1 area (left, lower). The number of neurons was significantly higher after rPTX3 treatment compared with vehicle treatment (right). (B) Neurons (NeuN, green) in the peri-injured cortex. DAPI (blue) was used to stain the nuclei (left). The number of neurons was significantly higher after rPTX3 treatment compared with vehicle treatment (right). Scale bars: 200 μm (hippocampus); 40 μm (cortex). **P < 0.01, ***P < 0.001. Data are expressed as the mean ± SEM (n = 6; one-way analysis of variance followed by Tukey post hoc test). DAPI: 4',6-Diamidino- 2-phenylindole; rPTX3: recombinant pentraxin 3.

Figure 4

rPTX3 treatment increases neurogenesis after traumatic brain injury. Neural progenitor cells and newly generated neurons were measured by immunostaining on day 21 after controlled cortical impact. (A) DCX (red) immunostaining in the ipsilateral hippocampus, with DAPI (blue) staining of the nuclei (left). The number of DCX-positive cells in the ipsilateral SGZ was significantly higher in the rPTX3 group compared with the control group (right). (B) DCX (red) and DAPI (blue) immunostaining in the ipsilateral SVZ (left). The number of DCX-positive cells in the ipsilateral SVZ was significantly higher in the rPTX3 group compared with the control group (right). (C) NeuN (green), BrdU (red), and DAPI (blue) immunostaining in the ipsilateral hippocampus (arrows; left). The number of NeuN/BrdU double-positive cells in the ipsilateral hippocampus was significantly higher in the rPTX3 group compared with the control group (right). (D) NeuN (green), BrdU (red), and DAPI (blue) immunostaining in the peri-injured cortex (arrow, left). The number of NeuN/BrdU double- positive cells in the peri-injured cortex was significantly higher in the rPTX3 group compared with the control group. Scale bars: 40 μm. ***P < 0.001. Data are expressed as the mean ± SEM (n = 6; one-way analysis of variance followed by Tukey post hoc test). BrdU: Bromodeoxyuridine; DAPI: 4',6-diamidino-2-phenylindole; DCX: doublecortin; NeuN: neuronal nuclear antigen; rPTX3: recombinant pentraxin 3; SGZ: subgranular zone; SVZ: subventricular zone.

Figure 5

rPTX3 promotes neurogenesis in vitro. Neurospheres were counted and immunostained at 7 days after passage. (A) Neurosphere formation (left). The number of neurospheres in neural progenitor cell cultures was significantly higher in the rPTX3 group compared with the control group (right). (B) BrdU (green) and DCX (red) immunostaining in neurospheres. DAPI (blue) represents the nucleus (left). The percentage of cells that were double- positive for DCX and BrdU was significantly higher in the rPTX3 group of neurospheres compared with the control group neurospheres (right). Scale bars: 100 μm. ***P < 0.001. Data are expressed as the mean ± SEM (n = 6; Student's t-test). BrdU: Bromodeoxyuridine; DAPI: 4',6-diamidino- 2-phenylindole; DCX: doublecortin; rPTX3: recombinant pentraxin 3.

Figure 6

rPTX3 promotes functional recovery after traumatic brain injury. Neurological function outcomes were measured after controlled cortical impact. (A, B) Motor-sensory functions, including NSS scores (A) and wire grip scores (B), were assessed 1 day before and on days 1, 3, 7, 14, and 21 after the controlled cortical impact. (C, D) Cognitive ability was assessed by the Morris water maze tests. In the navigation test, latency (C) and the time spent in the incorrect quadrant (D) were measured from days 16 to 20 after the controlled cortical impact. (E) The number of times the mice crossed the platform location in the probe trial test on day 21 after the controlled cortical impact. *P < 0.05, ***P < 0.001, vs. Control (Vehicle) group. Data are expressed as the mean ± SEM (n = 10; two-way repeated measures analysis of variance). NSS: Neurological severity scores; PTX3: pentraxin 3; rPTX3: recombinant pentraxin 3.