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Published in final edited form as: Prostaglandins Leukot Essent Fatty Acids. 2020 Dec 17;165:102227. doi: 10.1016/j.plefa.2020.102227

Palmitic Acid Methyl Ester Inhibits Cardiac Arrest-Induced Neuroinflammation and Mitochondrial Dysfunction

Celeste Yin-Chieh Wu 1, Alexandre Couto e Silva 2, Cristiane T Citadin 2, Garrett A Clemons 2, Christina H Acosta 2, Brianne A Knox 1, Mychal S Grames 3, Krista M Rodgers 1,2, Reggie Hui-Chao Lee 1,3, Hung Wen Lin 1,2,3
PMCID: PMC8174449  NIHMSID: NIHMS1661898  PMID: 33445063

Abstract

We previously discovered that palmitic acid methyl ester (PAME) is a potent vasodilator released from the sympathetic ganglion with vasoactive properties. Post-treatment with PAME can enhance cortical cerebral blood flow and functional learning and memory, while inhibiting neuronal cell death in the CA1 region of the hippocampus under pathological conditions (i.e. cerebral ischemia). Since mechanisms underlying PAME-mediated neuroprotection remain unclear, we investigated the possible neuroprotective mechanisms of PAME after 6 min of asphyxia cardiac arrest (ACA, an animal model of global cerebral ischemia). Our results from capillary-based immunoassay (for the detection of proteins) and cytokine array suggest that PAME (0.02 mg/kg) can decrease neuroinflammatory markers, such as ionized calcium binding adaptor molecule 1 (Iba1, a specific marker for microglia/macrophage activation) and inflammatory cytokines after cardiopulmonary resuscitation. Additionally, the mitochondrial oxygen consumption rate (OCR) and respiratory function in the hippocampal slices were restored following ACA (via Seahorse XF24 Extracellular Flux Analyzer) suggesting that PAME can ameliorate mitochondrial dysfunction. Finally, hippocampal protein arginine methyltransferase 1 (PRMT1) and PRMT8 are enhanced in the presence of PAME to suggest a possible pathway of methylated fatty acids to modulate arginine-based enzymatic methylation. Altogether, our findings suggest that PAME can provide neuroprotection in the presence of ACA to alleviate neuroinflammation and ameliorate mitochondrial dysfunction.

1. Introduction

Cardiopulmonary arrest (CA)-induced global ischemia remains to be an important clinical problem in the U.S. affecting more than 350,000 patients every year[1]. It causes delayed neuronal damage in the hippocampus and cortex[2], leading to death and disability with limited treatment options[3]. The major therapeutic challenge of CA is the hypoperfusion (indicative of depressed cerebral blood flow), which is believed to be the major cause of cognitive and memory dysfunction[4, 5]. The following inflammatory response is a crucial component of ischemic brain injury. Thus, development of novel therapies to alleviate hypoperfusion, decrease brain inflammation and cell death after CA are currently under investigation[4, 6].

Recently, studies have investigated the beneficial effects of fatty acids in cardiovascular health[7, 8]. The dietary consumption of omega-3 polyunsaturated fatty acids (n-3 PUFAs), particularly eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have been associated with the prevention of coronary artery diseases[9], reduction of depressive symptoms[10], and regulation of immune responses through inhibition of pro-inflammatory cytokines[11]. This is counterintuitive to saturated fatty acids (SFA), due to the increase in cardiovascular disease, obesity, and cancer. The desirable dietary ratio of polyunsaturated fatty acids to saturated fatty acids (P/S) is 1. P/S ratio of 1–1.5 in the diet is within a favorable range to reduce the risk of cardiovascular disease (CVD)[12]. However, actual dietary intakes of these fatty acids are lower than 1, particularly in Western countries[13, 14]. Thus, we seek to uncover endogenous compounds such as long chain fatty acids, which may be manipulated pharmacologically in order to produce more robust protective paradigms. We previously identified palmitic acid methyl ester (PAME), an endogenously released saturated fatty acid with potent vasodilatory properties. Interestingly, PAME is a 16-carbon saturated fatty acid derived from the sympathetic ganglia, which we have found to provide the neuroprotection following asphyxial cardiac arrest (ACA)[2, 15, 16].

PAME was found to enhance cortical cerebral blood flow and functional learning and memory, while inhibiting neuronal cell death in the CA1 region of the hippocampus after CA[2]. Therefore, we sought to understand the possible mechanism(s) of PAME-mediated neuroprotection via neuroinflammation, a critical effector of cellular damage following ischemia[17]. To assess the role of PAME in the neuroinflammatory response, a protein chip assay was used to evaluate cytokine/chemokine levels following PAME treatment. Global ischemia-induced learning and memory deficits has also been linked to neuronal metabolic dysfunction[18], thus we sought to evaluate the rescue of mitochondrial function in PAME-treated rats. According to previous publication, arginine analogs are critical for the endogenous release of PAME from superior cervical ganglion (SCG). The methylation of arginine is catalyzed by protein arginine methyltransferases (PRMTs), which is a post-translational modification involved in mRNA splicing, DNA repair, signal transduction, protein interaction, and transport [19, 20]. There are 10 PRMT isoforms and our main focus is on protein arginine methyltransferase 8 (PRMT8) since PRMT8 shares 80% sequence identity with PRMT1. Also, PRMT1 and PRMT8 are both involved in the epigenetic control of gene expression and the normal function of neurons [21, 22]. Our central hypothesis is that PAME provides neuroprotection following global ischemia through alleviation of post-ischemic neuroinflammation, rescue the function of mitochondria. This is correlated with protein arginine methyltransferase (PRMT) 1 and PRMT8 expression.

2. Materials and methods

2.1. Chemicals

PAME (P5177, Sigma-Aldrich, St. Louis, MO) was dissolved in 100 % ethanol (E7023, Sigma-Aldrich, St. Louis, MO) and then diluted with sterile saline to required concentrations. The injection vial was prewarmed at 37 °C for injection. For asphyxia cardiac arrest (ACA), animals received a single bolus injection of PAME at 0.02 mg/kg intraperitoneally (IP) immediately after ACA surgery. Details of chemicals and solutions used are found in previous publications[2, 15].

2.2. Animal preparation

All animal experimental procedures were approved by the Institutional Animal Care and Use Committee (Louisiana State University Health Sciences Center in Shreveport). Adult male Sprague-Dawley rats (300–350 grams) were acquired from Charles River Laboratories (Wilmington, MA) and housed for one week at the Louisiana State University Health Sciences Center Shreveport’s animal facilities. All rats were fasted overnight before surgery. Anesthesia was induced with 5 % isoflurane and a 30:70 mixture of O2 and N2O respectively, followed by endotracheal intubation. Normal respiration was maintained with a ventilator (VentElite Small Animal Ventilator Harvard Apparatus, Holliston, MA). After endotracheal intubation, isoflurane was reduced from 4 % to 1.5 %. The right femoral artery and vein were cannulated using a single-lumen (PE-50) catheter for the monitoring of mean arterial blood pressure (MABP), arterial blood gas analyses (pH, PO2, and pCO2), glucose assessment, and IV injection of drugs. The rats were immobilized with cisatracurium besylate (Nimbex, 0.4 mg/mL, IV every 10 mins) throughout the experiments. Head and body temperatures were continuously maintained around 37 °C using heating pads.

2.3. Asphyxial Cardiac Arrest (ACA)

After anesthesia, endotracheal intubation, and femoral artery/vein catheterization, nimbex (0.4 mg/mL, IV) was repeatedly administered every 10 mins throughout the surgery to immobilize the rat as needed. To induce ACA, asphyxia was initiated by disconnecting the ventilator from the endotracheal intubation tube. Cardiopulmonary resuscitation was initiated 6 mins after asphyxia by administering bolus IV injections of epinephrine (0.005 mg/kg) and sodium bicarbonate (1 mEq/kg) followed by mechanical ventilation with 100 % O2 at 80 breaths/min. We used the thumb, index, and middle fingers to administer manual chest compressions on the animal’s chest in a light circular motion on the x and z-axis (200/min) until the MABP reached 60 mmHg. PAME (0.02 mg/kg) was administered IP immediately after resuscitation. Blood gases were measured before and 10 mins after ACA[2, 4, 23].

2.4. Protein array for inflammatory cytokines

Frozen mouse hippocampal tissues (−80 °C) were lysed with ice cold T-PER® Tissue Protein Extraction Reagent (Thermo Scientific, Cat# 78510) with Halt™ Protease Inhibitor Cocktail (Thermo Scientific, Cat# 87785). The hippocampal tissue homogenates (1 μg/μL) were used to quantify inflammatory mediators via ELISA-based quantitative array platforms (RayBiotech, QAR-CAA-67). Each cytokine data point represents the mean of quadruplicates from individual mouse brain. The Quantibody® Rat Cytokine Array Kit includes 2 non-overlapping arrays that quantitatively measured 67 rat cytokines. The signal was visualized and scanned at 532 nm utilizing Cy3 dye and quantified. Cytokines that exhibited significantly differences (p ≤0.05) between groups were evaluated by unpaired t-test as appropriate with SPSS.

2.5. Capillary-based Immunoassay via ProteinSimple®

For Iba1 (ionized calcium-binding adapter molecule 1) expression analyses, the proteins were extracted from the hippocampus using T-PER® Tissue Protein Extraction Reagent (Thermo Scientific, Cat# 78510) with Halt™ Protease Inhibitor Cocktail (Thermo Scientific, Cat# 87785). The protein concentrations used for detection were 1 μg/μL for the total protein tissue lysates. Protein detection was performed using an automated capillary electrophoresis system (Wes and Compass software; ProteinSimple®, San Jose, CA, USA). Dilution of primary antibodies were used against the following proteins: Iba1 (1:50, GeneTex, GTX1000042), PRMT1 (1:20, Cell Signaling Technology, F339), PRMT8 (1:20, Cell Signaling Technology, F339) and β-actin (1:200, Cell Signaling Technology, #4970). Antibody targets were detected with an HRP-conjugated secondary anti-rabbit antibody. Specificity of PRMT1 and PRMT8 antibody are discussed in the supplementary material. Data are expressed and calculated by chemiluminescence. All data were normalized to β-actin.

2.6. Oxygen Consumption Analyses

Mitochondrial oxygen consumption rate (OCR) analysis was conducted via Seahorse XF24 Analyzer (Agilent Technologies) per manufacturer’s protocol. Whole brains after ACA were removed, dissected and placed into artificial cerebral spinal fluid buffer (aCSF) (120 mM NaCl, 3.5 mM KCl, 1.3 mM CaCl2, 1 mM MgCl2 hexahydrate, 0.4 mM KH2PO4, and 5 mM HEPES, with 10 mM glucose, and 1mg/mL of BSA added day of experiment, pH to 7.4 using 10 N NaOH). The whole brains were then sectioned on a coronal plane using a vibratome (Leica VT1000E, Leica, Wetzlar, Germany) at 200μm in thickness to prepare for OCR analysis. Brain slices were cut by 1-mm stainless steel biopsy punches then placed into XF Islet Capture Microplate (101122–100; Agilent Technologies, Santa Clara, CA) in aCSF. To measure OCR, the following inhibitors were used sequentially: 20 μg/mL Oligomycin (ATP-Synthase Inhibitor), 10 μM Carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (a mitochondrial membrane uncoupler), combined with 1 mM pyruvate, IV) 20 μM antimycin (complex III inhibitor). In this measurement, basal OCR <50 pmol/min resulted in erratic O2 readings, which caused unreliable OCR. Reserve capacity was calculated based on the average of all tissue sections. For further detailed concentrations and reagents please refer to previous publications[6].

2.7. Statistical analysis

Results were expressed as means ±SEM. Statistical analysis was evaluated by one-way ANOVA (Tukey’s HSD post-hoc test) or unpaired t-test as appropriate with SPSS (Chicago, IL). Differences with p ≤0.05 were considered statistically significant.

3. Results

3.1. PAME decreased ionized calcium binding adaptor molecule 1 (Iba1) protein levels after ACA

Hippocampal and cortical homogenates were used to measure Iba1 protein expression via ProteinSimple® capillary electrophoresis system. The Iba1 levels in hippocampus at 3 and 7 days after ACA were 1.5–2 times higher as compared to control. Post-treatment with PAME significantly decreased Iba1 expression at 3 and 7 days after ACA surgery (88.96 ± 11.53 % and 100.11 ± 11.53 %) as compared to ACA only groups (169.32 ± 12.88 % and 200.00 ± 25.60 %) (Fig. 1A). Similar to the hippocampus, results from the cortex show that ACA+PAMEpost (115.37 ± 22.78 %) can decrease ACA-enhanced Iba1 protein expression (223.74 ± 12.27 %) 7 days after ACA (Fig. 1B). Our results show that ACA+PAMEpost can decrease ACA-enhanced Iba-1 protein expression 3 and 7 days after ACA.

Fig 1. Post-treatment with PAME decreased ionized calcium binding adaptor molecule 1 (Iba1) after ACA.

Fig 1.

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Iba1 (24 kDa) (microglia/macrophage-specific calcium-binding) protein expression was measured (via ProteinSimple ® capillary electrophoresis system) in the hippocampus (A) and cortex (B). Iba1 protein levels (red bars) were enhanced in the presence of ACA, and decreased following post-treatment with PAME (0.02 mg/kg) (blue bars). Representative pseudo-blots are displayed below each bar graph. Results were expressed as mean ± SEM. *p≤0.05 as compared to control rats (white bar). #p≤0.05 as compared to 3 or 7 days after ACA, evaluated by one-way ANOVA with Tukey’s post-hoc test.

3.2. PAME decreased cytokines and chemokines after ACA

We utilized a protein chip assay to simultaneously examine multiple inflammatory cytokine/chemokine factors and adhesion molecules. Post-treatment with PAME significantly decreased the levels of pro-inflammatory cytokines and chemokines such as IFN-γ (63.79 ± 2.70 %), IL-1α (41.00 ± 4.26 %), IL-1β (71.19 ± 3.99 %), ICAM-1 (59.80 ± 4.25 %), and TIMP-1 (40.23 ± 6.73 %) 3 days after ACA as compared to ACA only (at baseline 100 %). Moreover, post-treatment with PAME also decreased pro-inflammatory cytokines and chemokines such as IL-6 (19.01± 8.85 %), 4–1BB (2.78± 2.02 %), CD48 (49.32± 7.50 %), and Activin A (36.46± 14.84 %) 7 days after ACA as compared to ACA only. IL-10, an anti-inflammatory cytokine, was significantly increased (212.24 ± 47.32 %) 7 days after ACA+PAMEpost as compared to ACA only. These results show that PAME can decrease overall inflammatory cytokines/chemokines expression at 3 and 7 days after ACA (Fig. 2), which further supports our Iba1 findings.

Fig 2. Post-treatment with PAME reduced inflammatory cytokines in the hippocampus 3 and 7 days after ACA.

Fig 2.

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(A) Post-treatment with PAME 3 days after ACA can decrease 5 analyzed cytokines as expressed in heat maps [green (low expression) to red (high expression)] via ELISA-based cytokine arrays. Post-treatment with PAME significantly reduced inflammatory cytokine levels in the hippocampus 3 days after ACA (blue bars) (B-F). (G) Post-treatment with PAME 7 days after ACA also can affect 5 cytokines as expressed in heat maps. Post-treatment with PAME significantly reduced inflammatory cytokine levels in the hippocampus 7 days after ACA (I-L), whereas the IL-10, anti-inflammatory cytokine, was significantly increased 7 days after ACA (H). Numbers in parentheses indicate the number of rat brain samples per group. Results were expressed as mean ± SEM. *p≤0.05 as compared to ACA only animals (red bar), evaluated by unpaired t test.

3.3. PAME alleviated hippocampal mitochondrial dysfunction 7 days after ACA

We previously showed that PAME can alleviate learning and memory deficits after cardiac arrest[2]. In order to investigate whether the PAME-induced enhancement of functional learning and memory might be related to mitochondrial function, we measured the mitochondria respiration from hippocampal tissue sections, in response to sequential pharmacological injections to quantify ATP production, ATP-linked respiration, proton leak-linked respiration, maximal respiration, reserve capacity, basal respiration, and coupling efficiency. Our findings demonstrate that after ACA, there was a significant reduction in reserve capacity (−3.23 ± 5.08 %) as compared to control animals (17.10 ± 1.49 %). Furthermore, treatment with PAME rescued mitochondrial reserve capacity after ACA (16.27 ± 4.04 %) (Fig. 3). These results demonstrate that ACA can decrease hippocampal mitochondrial respiration resilience against ischemia, altogether resulting in cellular stress. Oxidative stress within the CA1 region of the hippocampus can lead to cellular damage via depolarization of the mitochondrial inner membrane and impairment of ATP synthesis[24], ultimately resulting in hippocampal cell death, pro-inflammatory response, and learning/memory deficits. However, our findings demonstrate that the post-treatment of PAME can alleviate ACA-induced potential mitochondrial dysfunction.

Fig 3. Palmitic acid methyl ester (PAME) alleviated hippocampal mitochondrial dysfunction after ACA.

Fig 3.

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Oxygen consumption rate (OCR) was measured in hippocampal slices (200 μm) via a Seahorse XF24 analyzer. Mitochondrial reserve capacity was reduced 7 days after ACA while treatment with PAME (PAME, 0.02 mg/kg, IV, blue triangles) rescued mitochondrial reserve capacity. * represent p≤0.05, which is significantly different from control and PAME post-treatment groups, respectively. All data sets were evaluated by one-way ANOVA with Tukey’s post-hoc test and expressed as mean ± SEM (n= 5–7).

3.4. Increased expression of both PRMT1 and 8 is correlated with PAME-mediated neuroprotection and inhibited neuroinflammation

Cho JY et al., Yang X et al., and Cai S et al. have reported that PRMT regulates proinflammatory cytokines involved in the inflammatory response[25–27]. Here, we verified the potential roles of PRMT1 and PRMT8 in the PAME-mediated neuroprotection pathway. To confirm the expression of PRMT1 and PRMT8 in hippocampus, we examined protein expression by ProteinSimple® capillary electrophoresis system. As shown in Figure 4A, PRMT1 levels of hippocampus after 3 and 7 days ACA (0.095 ± 0.005 and 0.081 ± 0.006) were siginificantly decreased compared with the control group (0.140 ± 0.011). Post-treatment with PAME at 3 and 7 days after ACA surgery siginificantly enhanced PRMT1 levels (0.199 ± 0.013 and 0.253 ± 0.021) compared with the ACA only groups. Similarly, PRMT8 levels also decreased at 1, 3, and 7 days after ACA (0.057 ± 0.005, 0.048 ± 0.004, and 0.047 ± 0.003) as compared with the control group (0.081 ± 0.007), whereas abatement of PRMT expression were reversed after post-treatment of PAME after 3 and 7 days ACA (0.110 ± 0.005 and 0.144 ± 0.001) (Fig. 4B). These results show PAME can potentially reduce neuroinflammation via up-regulation of PRMT1 and PRMT8.

Fig 4. Post-treatment with PAME enhanced PRMT1 and PRMT8 expression in the hippocampus after ACA.

Fig 4.

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PRMT1 (~44 kDa, A) and PRMT8 (49 kDa, B) protein expression were measured (via ProteinSimple ® capillary electrophoresis system) in the hippocampus. The protein levels (red bars) were decreased after ACA, and enhanced with post-treatment with PAME (0.02 mg/kg) (blue bars). Representative pseudo-blots are displayed below each bar graph. Results were expressed as mean ± SEM. *p≤0.05 as compared to control animal (white bar). #p≤0.05 as compared to ACA groups, evaluated by one-way ANOVA with Tukey’s post-hoc test.

4. Discussion

We have previously demonstrated that palmitic acid methyl ester (PAME), a 16-carbon saturated fatty acid released from the sympathetic nervous system, can act as a potent vasodilator and enhance cerebral blood flow without affecting mean arterial pressure when administered systemically (IV)[2, 15]. These results demonstrate that there is a selective vasoactivity specifically in the brain. In addition, PAME post-treatment can revive asphyxial cardiac arrest-induced hypoperfusion 24 hrs after global ischemia, and protect hippocampal neurons while preserving functional learning/working memory 3 to 7 days after asphyxial cardiac arrest (ACA)[2, 16]. Our findings demonstrate that while neuroinflammatory responses were enhanced after ACA, as indicated by an increase in glial reactivity (Iba1 and pro-inflammatory cytokines expression), post-treatment with PAME reduced both the expression of Iba1 and pro-inflammatory cytokine levels (Figs. 1 and 2). Furthermore, we performed oxygen consumption rate measurements in hippocampal slices to demonstrate that PAME can rescue reserve capacity oxygen consumption rate, blunted with ACA in the mitochondria (Fig. 3F). We have also shown that PRMT 1 and 8 are enhanced in the presence of post-treatment with PAME. These results demonstrate that PAME can elevate PRMT1/8 protein levels to enhance arginine-based methylation and provide overall therapeutic benefits against ACA (Fig. 4). Based on our previous and current findings, enhanced PRMT levels correlate well with PAME-mediated neuroprotection (as shown previously), and the attenuation of neuroinflammatory mediators currently presented in this study. In the present study, our findings indicate that Iba1expression was enhanced 3 and 7 days after ACA in the hippocampus to demonstrate an increase in microglia/macrophage activation, which was attenuated with PAME post-treatment (Fig. 1). In addition, our findings also indicate that proinflammatory markers (i.e. IL-1β, IL-6, and IFN-γ) were enhanced after ACA, but decreased after post-treatment with PAME (Fig. 2)[28–30]. Here, our findings show that post-treatment of PAME can ameliorate ACA-induced neuroinflammation, specifically decreasing Iba1 levels and inflammatory cytokines (IFN-γ, IL-1α, IL-1β, ICAM-1, TIMP-1, IL-6, 4–1BB, CD48, and Activin A).

Other studies have recognized the role of the mitochondria as a regulator of inflammatory responses[31–33]. Therefore, we utilized the Seahorse XF24 analyzer to measure mitochondrial oxygen consumption rate in hippocampal slices of control and ACA-treated rats. Our results show that ACA decreased mitochondrial reserve capacity as compared to control and ACA+PAMEpost (Fig. 3B). In addition, our findings also indicate that PAME can enhance oxygen consumption in the hippocampus, otherwise decreased in the presence of ACA. Since respiration can be blunted in the presence of ischemia, the reserve capacity is an indication of how much “buffering” capacity the hippocampal slices can withstand in the presence of ischemia. In other words, even under ischemic conditions in the presence of PAME, oxygen consumption rate is similar to control (non-ischemia) levels to show proper mitochondria respiration. It is also important to note that our hippocampal slice technique developed from Fried et al., 2014[34] may be more physiologically relevant than the use of standard isolated mitochondria from tissues.

Our previous findings demonstrate that PAME, but not palmitic acid has vasodilatory properties[15], can enhance cerebral blood flow[2], and afford neuroprotection[16] under ischemic conditions. These results further show that the methylated form of palmitic acid is biologically active to cause more favorable outcomes after ischemia. This idea was fostered from the fact that arginine and its’ derivatives enhanced PAME release in the sympathetic neurons[15]. Additionally, our previous findings also show that in the presence of arginine derivatives, PAME release was enhanced in sympathetic neurons[15]. This led us to study the interactions of protein arginine methyltransferases (PRMTs) as it relates to PAME, due to the fact that PRMTs can methylate different species in the presence of arginine and its derivatives. PRMT8 is primarily expressed in the central nervous system [21, 22]. In fact, PRMT8 is localized to the cellular membrane via myristoylation located on the N-terminus and shares 80% sequence homology to PRMT1, which has major methylation activity [35, 36]. PRMT1 and PRMT8 both have roles in regulating epigenetic gene expression and serve as post-translational modifiers of proteins (e.g., sodium ion channels) [37–39]. Furthermore, co-expression of PRMT8 and the sodium channel 1.2 led to an increase in sodium currents, serving as an indicator that arginine-based methylation modulates neuronal excitability [40]. Most of the current literature concerning PRMT8 has been attributed to the involvement of neuronal development [41] implicated in dendritic spine maturation and modulation of the actin cytoskeleton in the absence of cellular stress [42, 43]. We found that hippocampal PRMT1 and PRMT8 are enhanced in the presence of PAME showing that PAME can also drive PRMT1 and PRMT8 protein expression. PRMT1 is a more ubiquitous methylator while PRMT8 is more exclusive to the central nervous system[35]. Here, our data show PRMT1 and 8 play crucial role in the context of cellular stress, the importance of PAME-mediated anti-inflammation, and neuroprotection. To our knowledge, this was the first study to correlate fatty acids to PRMTs.

5. Conclusion

In conclusion, CA remains one of the leading causes of death in the U.S. showing that lack of novel therapies to efficiently enhance survival rate and functional outcomes is urgently needed. Here, we report that post-treatment with PAME can decrease Iba1 and pro-inflammatory cytokine/chemokine levels in the rat hippocampus, while conserving mitochondrial reserve capacity. In addition, PRMT1 and 8 enzymes are enhanced in the presence of PAME to demonstrate a possible mechanistic pathway for the methylation of fatty acids. Therefore, more studies into the metabolic consequences of PAME in the context of cerebral ischemia are necessary. Our study will provide further understanding of PAME and PRMT enzymes as beneficial treatments/targets against cerebral ischemia.

Supplementary Material

1

Acknowledgements

This study was supported by grants from the NIH/NINDS R01-NS096225 and the AHA 19TPA34850047, 19CDA34660032, 19POST34380784, 19PRE34390909, and Louisiana State University Research Council.

Footnotes

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