Multiple mechanisms underlying neuroprotection by secretory phospholipase A2 preconditioning in a surgically induced brain injury rat model

Abstract

Background: Intra-operative bleeding, post-operative brain edema and neuroinflammation are major complications in patients with surgical brain injury (SBI). Phospholipase A2 (PLA2) is the upstream enzyme which initiates the PLA2, 5-lipoxygenase (5-LOX) and leukotriene B4 (LTB4) inflammatory pathway. We hypothesized PLA2preconditioning (PPC) prior to SBI can activate endogenous anti-inflammatory responses to protect against SBI. This study evaluated if PPC can ameliorate neurosurgical complications and elucidated PPC-mediated possible protective mechanisms in a rat SBI model.

Methods: Total 105 adult male Sprague Dawley rats were used for this study. SBI was induced by partial resection of the right frontal lobe. PLA2 or 0.9% NaCl was injected via rats' tail vein for 3 consecutive days prior to SBI. For mechanism study, a selective PLA2 inhibitor, Manoalide and 5-LOX inhibitor, Zileuton were injected intravenously with PPC to elucidate the role of PLA2 and 5-LOX in PPC-mediated anti-inflammatory effects. Brain water content (BWC) and lung water content, neurological tests, ELISA, western blot, immunohistochemistry, white blood cells (WBC) count, and spectrophotometric assay for intra-operative hemorrhage volume were evaluated.

Results: First, PPC reduced brain water content, intra-operative bleeding, and improved neurological function after SBI. Second, PPC decreased 5-LOX expression and brain leukocyte infiltration, while increasing glial fibrillary acidic protein (GFAP) expression in the peri-resection brain tissue after SBI. Third, PPC induced peripheral inflammation represented by mild pulmonary inflammation and increased peripheral blood WBC count and LTB4 level. Lastly, PPC increased blood glucose concentration and glucocorticoid levels after SBI. In addition, PPC mediated above-mentioned changes were partially reversed by administration of PLA2 inhibitor, Manoalide and 5-LOX inhibitor, Zileuton.

Conclusions: PPC conferred neuroprotection against SBI via multi-target involvement induced anti-inflammatory mechanisms.

Keywords: Brain edema; Intra-operative hemorrhage; Neuroinflammation; Preconditioning; Surgical brain injury; sPLA2.

Fig. 1

Experimental design and animal groups. Experiment 1: neurological tests were evaluated at 24h and 72h after surgery following which the animals were euthanized to collect brain samples for brain water content (BWC) measurement (n=6/group/time point). Experiment 2: animals were euthanized at 24h after surgery. Peripheral blood sample was collected during sacrifice for WBC count and LTB4 assay (n=8/group), and brain samples were harvested from the same animals for western blot (n=6/group) and immunohistochemistry (n=2/group). Experiment 3: neurological tests were evaluated at 24h following which animals were euthanized. Peripheral blood sample was collected during sacrifice for WBC count and LTB4 assay (n=8/group), after which brain samples were harvested from the same animals for western blot (n=6/group). Samples for SPC+SBI were shared with Experiment 2. Vehicle refers to ethanol. SBI, surgical brain injury; SPC, saline preconditioning, PPC, PLA2 preconditioning; BWC, brain water content, IHC, immunohistochemistry; WBC, white blood cells; LTB4, leukotriene B4.

Fig. 2

Effects of PPC on brain water content (BWC) and neurobehavioral performance at 24h and 72h post SBI. (A and B) PPC+SBI group had significantly decreased BWC in the right frontal lobe compared to SPC+SBI group at 24h and 72h post surgery. (C and D) Modified Garcia score was increased significantly in PPC+SBI group compared to SPC+SBI at 24h and 72h after SBI. (E and F). Beam balance score showed no significant differences between PPC+SBI and SPC+SBI groups. Data are expressed as mean±SEM. n=6/group. ANOVA, SNK. *P<0.05 vs Sham; Δp<0.05 vs SPC+SBI; RF=Right frontal lobe, LF=Left frontal lobe, RP=Right parietal lobe, LP=Left parietal lobe, C=cerebellum, BS= Brainstem.

Fig. 3

PPC induced mild pulmonary inflammation after SBI. (A)Water content of the lungs showed no significant difference within Sham, SPC+SBI and PPC+SBI groups at 24h and 72h after surgery. Data are expressed as mean±SEM. n=6/group. ANOVA, SNK. (B)Representative pictures show gross lung morphology in Sham, SPC+SBI and PPC+SBI groups at 24h after surgery. Gross lung morphology was similar in the three groups at 24h post surgery. n=6/group. (C) H&E staining of lung tissues demonstrated no obvious morphological changes in the three groups at 24h post surgery. Scale bar=100μm. n=2/group. Data not quantified. (D) PPC+SBI group lung samples showed that MPO-positive leukocytes co-localized with 5-LOX and (E) Some MPO-positive leukocytes were TUNEL-positive at 24h post SBI. Scale bar=50μm (D and E). n=2/group. Data not quantified (D and E).

Fig. 4

PPC increased peripheral blood WBC count measured 24h post SBI. Peripheral WBC count was increased in PPC+SBI compared with Sham and SPC+SBI groups. Data are expressed as mean±SEM. n=8/group. ANOVA, SNK. *p<0.05 vs Sham, Δp<0.05 vs SPC+SBI.

Fig. 5

PPC effects on blood glucose, glucocorticoid levels and intra-operative hemorrhagic volume at 24h post surgery. (A)Significant elevation of blood glucose was observed in PPC+SBI group compared to SPC+SBI group. There was no difference between Sham and SPC+SBI groups.(B)PPC+SBI group had significantly increased blood glucocorticoid concentration compared to SPC+SBI rats. There was no significant difference between Sham and SPC+SBI rats. (C) PPC+SBI group had significantly decreased intra-operative hemorrhagic volume compared to SPC+SBI rats. Data are expressed as mean±SEM. n =8/group. ANOVA, SNK.*p<0.05 vs Sham, Δp<0.05 vs SPC+SBI.

Fig. 6

Subcellular distribution of 5-LOX in the ipsilateral hemisphere at 24h post SBI. 5-LOX is expressed mainly in NeuN-labeled neurons (A) and GFAP-labeled astrocytes (B) but not in Iba1-labeled microglia (C) 24h after SBI. Arrows indicate colocalization of 5-LOX with NeuN (A) and GFAP with Iba1 (B). Sham control group did not show 5-LOX staining in the right frontal region at 24h (D). Scale bar=25μm(A-C)and 50μm(D). n=2/group. Data not quantified.

Fig. 7

PPC effects on the expression of cerebral 5-LOX, GFAP and MPO at 24h after surgery. (A) Immunofluorescence pictures showing the expression of 5-LOX, GFAP and MPO in Sham, SPC+SBI and PPC+SBI rats. Scale bar=50μm. n=2/group. Data not quantified. Representative western blot bands (B) and quantitative analysis (C) showed the expression of 5-LOX and MPO decreased in PPC+SBI rats compared to SPC+SBI rats and GFAP expression increased in PPC+SBI rats compared to SPC+SBI rats. Data are expressed as mean±SEM. n=6/group. ANOVA, SNK. *p<0.05 vs Sham; Δp<0.05 vs SPC+SBI.

Fig. 8

Effects of Manoalide and Zileuton on peripheral blood WBC count, LTB4 level and neurological scores 24h after surgery. Compared to SPC+SBI rats, PPC+SBI group had significantly increased WBC count (A) and blood LTB4 level (B), which was reversed with Manoalide and Zileuton. PPC+SBI group showed improved modified Garcia neurological which was reversed with Manoalide and Zileuton (C). Beam balance scores was not significantly different between groups (D). Data are expressed as mean±SEM. n=8/group. ANOVA, SNK.*p<0.05 vs SPC+SBI; Δp<0.05 vs PPC+SBI.

Fig. 9

Manoalide and Zileuton reversed PPC-induced anti-inflammatory effects at 24h after surgery. (A) Representative pictures of western blot bands and (B)Quantitative analysis of the bands indicated that PPC+SBI had significantly reduced levels of 5-LOX and MPO in the peri-resection brain tissue compared to SPC+SBI rats, which were reversed by Manoalide and Zileuton. Data are expressed as mean±SEM. n=6/group. ANOVA, SNK.*p<0.05 vs SPC+SBI; Δp <0.05 vs PPC+SBI.