Neuroprotection after traumatic brain injury in heat-acclimated mice involves induced neurogenesis and activation of angiotensin receptor type 2 signaling - PubMed (original) (raw)

Neuroprotection after traumatic brain injury in heat-acclimated mice involves induced neurogenesis and activation of angiotensin receptor type 2 signaling

Gali Umschweif et al. J Cereb Blood Flow Metab. 2014 Aug.

Abstract

Long-term exposure of mice to mild heat (34°C±1°C) confers neuroprotection against traumatic brain injury (TBI); however, the underling mechanisms are not fully understood. Heat acclimation (HA) increases hypothalamic angiotensin II receptor type 2 (AT2) expression and hypothalamic neurogenesis. Accumulating data suggest that activation of the brain AT2 receptor confers protection against several types of brain pathologies, including ischemia, a hallmark of the secondary injury occurring following TBI. As AT2 activates the same pro-survival pathways involved in HA-mediated neuroprotection (e.g., Akt phosphorylation, hypoxia-inducible factor 1α (HIF-1α), and brain-derived neurotrophic factor (BDNF)), we examined the role of AT2 in HA-mediated neuroprotection after TBI. Using an AT2-specific antagonist PD123319, we found that the improvements in motor and cognitive recovery as well as reduced lesion volume and neurogenesis seen in HA mice were all diminished by AT2 inhibition, whereas no significant alternations were observed in control mice. We also found that nerve growth factor/tropomyosin-related kinase receptor A (TrkA), BDNF/TrkB, and HIF-1α pathways are upregulated by HA and inhibited on PD123319 administration, suggesting that these pathways play a role in AT2 signaling in HA mice. In conclusion, AT2 is involved in HA-mediated neuroprotection, and AT2 activation may be protective and should be considered a novel drug target in the treatment of TBI patients.

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Figures

Figure 1

Figure 1

Heat acclimation (HA)-mediated neuroprotection and cognitive performance are compromised by angiotensin receptor type 2 blockade after traumatic brain injury (TBI). PD123319 (PD, 10 mg/kg/day) or saline-treated mice were subjected to TBI, and neurobehavioral outcome was evaluated by the Neurological Severity Score (NSS) at 1 hour post injury to assess initial disability and at multiple time points thereafter. PD treatment eliminated the statistically significant improvement in neurobehavioral outcome of HA mice (A; *P<0.05; **P<0.01 HA treated with saline versus normothermic (NT) treated with saline, determined by two-way analysis of variance for repeated measures, _n_=9 to 10 per group). For the sake of clarity, maximal error bars of the groups treated with PD123319 are presented on the right end of the representative line. In the box plots, mice were subjected to the novel object recognition test (NORT) 3 days (B) and 30 days (C) after TBI. The graph shows the median (band), 25th and 75th quantiles (bottom and top of box), sample minimum and maximum (whiskers), and outliers (dots). The longer exploration time of the novel object of HA mice decreased after PD123319 (PD, 10 mg/kg/day) treatment. *P<0.05 HA treated with saline versus NT treated with saline; #P<0.05 HA treated with saline versus HA treated with PD123319, determined by Mann–Whitney, _n_=9 to 10 per group.

Figure 2

Figure 2

Heat acclimation (HA)-mediated reduced lesion volume is eliminated by angiotensin receptor type 2 blockade after traumatic brain injury (TBI). Mice were killed 42 days after TBI, and evenly separated 10-_μ_m slices were stained with Giemsa. (A) The graph shows the median (band), 25th and 75th quantiles (bottom and top of box), sample minimum and maximum (whiskers), and outliers (dots). (B) The reduced lesion volume measured in HA mice (panels a–c) was abolished by PD123319 treatment (panels d–f, PD123319, 10 mg/kg/day). *P<0.05 HA treated with saline versus normothermic (NT) treated with saline (panels g–i); #P<0.05 HA treated with saline versus HA treated with PD123319, as determined by Mann–Whitney (_n_=6 per group).

Figure 3

Figure 3

Angiotensin receptor type 2 (AT2) blockade decreases protein levels of AT2 and hypoxia-inducible factor 1_α_ (HIF-1_α_) in heat-acclimated (HA) mice. Mice were subjected to traumatic brain injury (TBI) after treatment with saline or PD123319 (PD, 10 mg/kg/day), and killed at 6, 24, or 72 hours post TBI. (A) HA did not alter AT2 levels; however, PD123319 treatment lowered AT2 levels in HA mice. (B) Elevated HIF-1_α_ levels were measured in HA mice and were reduced after PD123319 treatment. *P<0.05 between HA treated with saline versus normothermic (NT) treated with saline; **P<0.05 between HA treated with PD123319 versus NT treated with PD123319; #P<0.05 HA treated with saline versus HA treated with PD123319, determined by two-way analysis of variance followed by Tukey's test, _n_=6 per group.

Figure 4

Figure 4

Angiotensin receptor type 2 is involved in heat acclimation (HA)-mediated enhanced neurotrophin signaling. Mice were subjected to traumatic brain injury (TBI) after treatment with saline or PD123319 (PD, 10 mg/kg/day), and were killed at 6, 24, or 72 hours post TBI. HA elevated levels of tropomyosin-related kinase receptor A (TrkA, A) as well as its endogenous ligand, nerve growth factor (NGF). PD123319 treatment decreased NGF levels in HA mice (B). Elevated TrkB levels were seen in HA mice and were reduced after PD123319 treatment in both normothermic (NT) and HA mice (C). HA induced brain-derived neurotrophic factor (BDNF) levels, whereas PD123319 treatment reduced BDNF levels, in both NT and HA mice (D). *P<0.05 HA treated with saline versus NT treated with saline; #P<0.05 mice treated with saline versus mice treated with PD123319 of the same group (NT/HA), determined by two-way analysis of variance followed by Tukey's test, _n_=6 per group.

Figure 5

Figure 5

Angiotensin receptor type 2 is involved in heat acclimation (HA)-mediated enhanced neurogenesis in the subventricular zone (SVZ). Mice were subjected to traumatic brain injury (TBI) after treatment with saline or PD123319 (PD, 10 mg/kg/day) for 3 days, and killed 42 days post TBI. Evenly sliced 10-_μ_m sections were incubated with anti-NeuN for mature neurons and with anti-BrdU (anti-5-bromo-2-deoxyuridine) for newborn cells. (A) HA induced neurogenesis in the SVZ (i, j) and PD123319 (PD, 10 mg/kg/day) treatment blocked the observed neurogenesis (m–p). PD treatment (e–h) did not reduce neurogenesis in normothermic (NT) mice measured after saline treatment (a–d). Stereological quantification of the fields was used to count the number of BrdU-positive cells (BrdU+). Results show the average number of BrdU+ per field (B), and the average number of double-positive cells for NeuN and for BrdU (BrdU+/NeuN+) per field (C). *P<0.01 HA treated with saline versus NT treated with saline; #P<0.01 HA mice treated with saline versus HA mice treated with PD123319, determined by two-way analysis of variance followed by Tukey's test, _n_=6 per group.

Figure 6

Figure 6

Angiotensin receptor type 2 is involved in heat acclimation (HA)-mediated enhanced neurogenesis in the dentate gyrus. Mice were subjected to traumatic brain injury (TBI) and treated with saline or PD123319 (PD, 10 mg/kg/day) for 3 days, and killed 42 days post TBI. Evenly sliced 10-_μ_m sections were incubated with anti-NeuN for mature neurons and with anti-BrdU (anti-5-bromo-2-deoxyuridine) for newborn cells. (A) HA induced neurogenesis in the dentate gyrus subgranular zone (i, j) and PD123319 (PD, 10 mg/kg/day) treatment blocked the observed neurogenesis (m–p). PD treatment (e–h) did not reduce neurogenesis in normothermic (NT) mice (a–d). Stereological quantification of the fields was used to count the number of BrdU-positive cells (BrdU+). Results show the average number of BrdU+ per field (B), and the average number of double-positive cells for NeuN and for BrdU (BrdU+/NeuN+) per field (C). *P<0.05 HA treated with saline versus NT treated with saline; #P<0.05 HA mice treated with saline versus HA mice treated with PD123319, determined by two-way analysis of variance followed by Tukey's test, _n_=6 per group.

Figure 7

Figure 7

Angiotensin receptor type 2 is involved in heat acclimation (HA)-mediated enhanced neurogenesis in the injury region (inj). Mice were subjected to traumatic brain injury (TBI) and treated with saline or PD123319 (PD, 10 mg/kg/day) for 3 days, and killed 42 days post TBI. Evenly sliced 10-_μ_m sections were incubated with anti-NeuN for mature neurons and with anti-BrdU (anti-5-bromo-2-deoxyuridine) for newborn cells. (A) HA induced neurogenesis in the injury region (i, j) and PD123319 (PD, 10 mg/kg/day) treatment blocked the observed neurogenesis (m–p). PD treatment (e–h) did not reduce neurogenesis in normothermic (NT) mice (a–d). Stereological quantification of the fields was used to count the number of BrdU-positive cells (BrdU+). Results show the average number of BrdU+ per field (B), and the average number of double-positive cells for NeuN and for BrdU (BrdU+/NeuN+) per field (C). *P<0.01 HA treated with saline versus NT treated with saline; #P<0.01 HA mice treated with saline versus HA mice treated with PD123319, determined by two-way analysis of variance followed by Tukey's test, _n_=6 per group.

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