Lactate potentiates angiogenesis and neurogenesis in experimental intracerebral hemorrhage

Abstract

Lactate accumulation has been observed in the brain with intracerebral hemorrhage (ICH). However, the outcome of lactate accumulation has not been well characterized. Here, we report that lactate accumulation contributes to angiogenesis and neurogenesis in ICH. In the first set of the experiment, a rat model of ICH was induced by injecting collagenase into the brain. The effects of lactate accumulation on the neurological function, apoptosis, and numbers of newborn endothelial cells and neurons, as well as the proliferation-associated signaling pathway, were evaluated in the rat brain. In the second set, exogenous L-lactate was infused into intact rat brains so that its effects could be further assessed. Following ICH, lactate accumulated around the hematoma; the numbers of PCNA⁺/vWF⁺ nuclei and PCNA⁺/DCX⁺ cells were significantly increased compared with the numbers in the Sham group. Moreover, ICH induced translocation of nuclear factor-kappa B (NF-κB) p65 into the nucleus, resulting in a notable upregulation of VEGF and bFGF mRNAs and proteins compared with the levels in the Sham controls. Administration of a lactate dehydrogenase inhibitor dramatically inhibited these effects, decreased the vascular density, and aggravated neurological severity scores and apoptosis after ICH. After exogenous L-lactate infusion, the numbers of PCNA⁺/vWF⁺ nuclei and PCNA⁺/DCX⁺ cells were strikingly increased compared with the numbers in the Sham controls. In addition, lactate facilitated NF-κB translocation to induce increased transcription of VEGF and bFGF. Co-infusion with an NF-κB inhibitor significantly inhibited these effects. These data suggest that lactate potentiates angiogenesis and neurogenesis by activating the NF-κB signaling pathway following ICH.

Fig. 1

a Lactate production was significantly increased around the hematoma after ICH. OXA markedly lowered the lactate level (n = 5). b OXA-treated rats showed a significantly increased mNSS compared with that of the ICH group (n = 8). c Nissl staining showed that many neurons in the ICH group were damaged (arrows without tails); the number of surviving neurons (arrows with tails) was markedly reduced. In the OXA group, far fewer surviving cells were observed (n = 5). d TUNEL-positive cells markedly increased in abundance after ICH. In particular, the number of TUNEL-positive cells was significantly increased in the OXA-treated group after ICH (n = 5). ICH intracerebral hemorrhage, OXA oxamate. *p < 0.05 and **p < 0.01. Scale bar = 50 μm

Fig. 2

a Newborn nuclei in vWF⁺ vessels were present around the hematoma after ICH; OXA dramatically reduced PCNA⁺ nuclei in ECs. b In the ICH group, the brain vasculature was deformed and blood flow was obstructed. OXA further diminished the vascular density after ICH. c,d PCNA⁺/DCX⁺ cells were more prevalent after ICH than in the Sham group. OXA infusion notably decreased the numbers of PCNA⁺/DCX⁺ cells after ICH. ICH intracerebral hemorrhage; OXA oxamate. *p < 0.05 and **p < 0.01. n = 5. Scale bar = 100 μm

Fig. 3

a,b Degradation and phosphorylation of IκBα occurred after ICH. OXA dramatically inhibited this effect. c Nuclear translocation of NF-κB p65 was significantly increased after ICH. This effect was significantly blocked by OXA. d Immunohistochemical analysis showed that NF-κB p65 was mainly retained in the cytoplasm in the Sham group, but some of the NF-κB p65 was transferred into the nucleus after ICH. ICH intracerebral hemorrhage, OXA oxamate. *p < 0.05 and **p < 0.01. n = 5. Scale bar = 50 μm

Fig. 4

a Levels of VEGF and bFGF mRNAs were dramatically decreased in the OXA-treated group after ICH. b Western blot analysis showed that the expression levels of VEGF and bFGF were significantly upregulated after ICH. OXA suppressed this upregulation. c After ICH, many VEGF⁺ microvessels and bFGF⁺ cells were detected around the hematoma. d Immunofluorescent double labeling confirmed that VEGF was localized in vWF-positive vessels and that bFGF was localized to NeuN-positive cells. ICH intracerebral hemorrhage, OXA oxamate. *p < 0.05 and **p < 0.01. n = 5. Scale bar = 100 μm

Fig. 5

a,b IκBα was degradated and phosphorylated after l-lactate infusion, and NF-κB p65 protein translocated into the nucleus. c The expression levels of VEGF and bFGF were strikingly increased after l-lactate infusion. BAY blocked these effects. d l-lactate significantly increased the expression of PCNA⁺/vWF⁺ nuclei and PCNA⁺/DCX⁺ cells. BAY blocked these effects. Lac l-lactate, BAY BAY11-7082. *p < 0.05 and **p < 0.01. n = 5. Scale bar = 100 μm

Fig. 6

Lactate potentiates angiogenesis and neurogenesis by activating the NF-κB signaling pathway following ICH