Lithium modifies brain arachidonic and docosahexaenoic metabolism in rat lipopolysaccharide model of neuroinflammation.

Neuroinflammation, caused by 6 days of intracerebroventricular infusion of a low dose of lipopolysaccharide (LPS; 0.5 ng/h), stimulates brain arachidonic acid (AA) metabolism in rats, but 6 weeks of lithium pretreatment reduces this effect. To further understand this action of lithium, we measured concentrations of eicosanoids and docosanoids generated from AA and docosahexaenoic acid (DHA), respectively, in high-energy microwaved rat brain using LC/MS/MS and two doses of LPS. In rats fed a lithium-free diet, low (0.5 ng/h)- or high (250 ng/h)-dose LPS compared with artificial cerebrospinal fluid increased brain unesterified AA and prostaglandin E(2) concentrations and activities of AA-selective Ca(2+)-dependent cytosolic phospholipase A(2) (cPLA(2))-IV and Ca(2+)-dependent secretory sPLA(2). LiCl feeding prevented these increments. Lithium had a significant main effect by increasing brain concentrations of lipoxygenase-derived AA metabolites, 5- hydroxyeicosatetraenoic acid (HETE), 5-oxo-eicosatetranoic acid, and 17-hydroxy-DHA by 1.8-, 4.3- and 1.9-fold compared with control diet. Lithium also increased 15-HETE in high-dose LPS-infused rats. Ca(2+)-independent iPLA(2)-VI activity and unesterified DHA and docosapentaenoic acid (22:5n-3) concentrations were unaffected by LPS or lithium. This study demonstrates, for the first time, that lithium can increase brain 17-hydroxy-DHA formation, indicating a new and potentially important therapeutic action of lithium.

The goal of this study was to further investigate the interaction between chronic lithium and neuroinfl ammation by measuring concentrations of unesterifi ed polyunsaturated fatty acids and some of their metabolites in high-energy microwaved brain of rats fed LiCl chronically, using RP-HPLC/MS/MS as described in our ischemia study ( 29 ). We quantifi ed concentrations of unesterifi ed AA, DHA, docosapentaenoic acid (DPA; 22:5n-3), 17-OH-DHA, PGE 2 , TXB 2 , 5-, 12-and 15-hydroxyeicosatetraenoic acids (HETEs), and 5-oxo-eicosatetraenoic acid (5-oxo-ETE) in brains from rats subjected to 6 days of intracerebroventricular infusion with a high (250 ng/h) or low (0.5 ng/h) dose of LPS. The rats had been fed a control lithium-free or a therapeutically relevant LiCl diet for 36 days prior to LPS infusion (total diet duration 42 days) ( 25 ). Whole brain activities of cPLA 2 -IV, iPLA 2 -VI, sPLA 2 , and 15-LOX-2 protein levels were measured. Briefl y, we confi rmed previous observations regarding the effect of lithium on AA and PGE 2 in a model of neuroinfl ammation with the RP-HPLC/MS/MS technique and extended the list of analyzed metabolites, including 5-, 12-, and 15-HETE and 17-OH-DHA. We also found that the brain concentration of 17-OH-DHA, the precursor of several antiinfl ammatory mediators known as resolvins, was increased in LiCl-fed rats infused with artifi cial cerebrospinal fl uid (aCSF) and LPS, suggesting a new benefi cial mechanism of action of lithium in bipolar disorder as an antiinfl ammatory agent.

Animals
All procedures were performed under a protocol (no. 06-026) approved by the Animal Care and Use Committee of Eunice Kennedy Shriver National Institute of Child Health and Human Development, in accordance with the National Institutes of Health guidelines on the care and use of laboratory animals. Two-monthold male Fischer F344 rats (Taconic Farms, Rockville, MD) were housed in a facility with a 12/12, light/dark cycle. One group of rats was fed ad libitum Purina 5001 chow containing 1.70 g LiCl/ kg (low LiCl) for 4 weeks, followed by chow containing 2.55 g LiCl/kg (high LiCl) for 2 weeks (Harlan Telkad, Madison, WI) ( 25 ). This regimen produces plasma and brain lithium concentrations of about 0.7 mM, therapeutically relevant to bipolar disorder ( 19,33 ). Control rats were fed lithium-free Purina 5001 chow for 6 weeks. Water and NaCl solution (0.45 M) were available ad libitum to both groups.

Total fatty acid concentrations in control and LiCl diets
To analyze each diet, total lipids were extracted ( 34 ) from random 0.7-0.8 g samples (n = 4). An aliquot of total lipid extract was methylated with 1% H 2 SO 4 -methanol for 3 h at 70°C. Fatty acid methyl esters were then separated and quantifi ed by gasliquid chromatography. Before the sample was methylated, di-17:0 choline glycerophospholipid was added as an internal standard.

Surgery
Rats were anesthetized and an indwelling cerebroventricular cannula was fi xed in place as previously described ( 21,23,25 ). Artifi cial cerebrospinal fl uid (aCSF) or LPS (Sigma, Saint Louis, docosanoids, including resolvins, docosatrienes, and neuroprotectins. These novel oxygenated products of DHA were identifi ed in resolving infl ammatory exudates ( 9 ) and similar chemical structures were elucidated in tissues rich in DHA such as the brain (10)(11)(12). Hence, the terms resolvin (resolution phase interaction product) and docosatriene were introduced, because they displayed potent antiinfl ammatory and immunoregulatory properties. The enzymatic conversion of DHA to docosanoids has not been fully characterized but appears to involve an initial conversion of DHA to 17 S -hydroxy-DHA (17-OH-DHA) by a 15lipoxygenase (LOX)-like enzyme and further conversion to resolvins D via epoxide intermediates ( 13 ). So far, only isolated soybean and potato 15-LOX and porcine 12-LOX have been shown to convert DHA to 17-OH-DHA in vitro ( 10,14,15 ). In addition, the oxygenation of DHA to 17-OH-DHA can be mediated by nonenzymatic autoxidation ( 16 ).
Lithium has been used to treat bipolar disorder for over 50 years and remains the most common treatment for its manic phase ( 17,18 ). While lithium's mechanism of action is not agreed on, recent animal studies suggest that lithium downregulates the brain AA cascade by decreasing AA turnover within brain phospholipids ( 19 ) and the prostaglandin E 2 (PGE 2 ) concentration ( 20 ). To study the effects of lithium on the brain AA and DHA cascades during neuroinfl ammation, we used an animal model of neuroinfl ammation. In rats, neuroinfl ammation can be produced by chronic infusion of bacterial lipopolysaccharide (LPS) into the fourth cerebral ventricle ( 21 ). A 6 day infusion of high-dose LPS (250 ng/h) increases activated microglia in the thalamus ( 22 ). A lower LPS dose (0.5 or 1 ng/h) infused for 6 or 30 days produces behavioral defi cits, induces amyloid deposits, and activates microglia and astrocytes ( 23,24 ). We reported that a 6 day infusion of the low dose also increases markers of the brain AA metabolic cascade: activities of cytosolic AA-selective Ca 2+ -dependent PLA 2 (cPLA 2 ) and secretory PLA 2 (sPLA 2 ), turnover of AA in phospholipids, and concentrations of unesterifi ed AA and its PGE 2 and thromboxane B 2 (TXB 2 ) metabolites measured by ELISA or gas-liquid chromatography on highenergy microwaved brain tissue ( 23,25 ). Feeding LiCl to rats for 6 weeks to produce plasma and brain lithium concentrations therapeutically relevant to bipolar disorder prevented many of these LPS-induced increments ( 25 ). The LPS infusion did not change the brain unesterifi ed DHA concentration ( 23 ), DHA turnover in brain phospholipids ( 26 ), or activity of Ca 2+ -independent PLA 2 (iPLA 2 ), which is selective for DHA ( 23,27 ).
Reverse phase (RP) HPLC/MS/MS has emerged as one of the most specifi c and sensitive approaches used in the analysis of lipid mediators in biological samples ( 28 ). This method has been validated for quantifying concentrations of unesterifi ed fatty acids and their metabolites in rodent brains that have been subjected to high-energy headfocused microwaving to stop lipid metabolism and limit postmortem alterations ( 29,30 ). Others and we have demonstrated that such radiation is essential for measuring accurate brain concentrations of unesterifi ed fatty acids, eicosanoids, and anandamide ( 31 ). Indeed, during global

Statistical analysis
A two-way ANOVA, comparing diet (LiCl vs. control) with infusion (LPS vs. aCSF) was performed for body weight loss, brain lipids, and PLA 2 activities using SPSS 16.0. When LiCl × LPS interactions were statistically insignifi cant, probabilities of main effects of LiCl and LPS were reported. When interactions were statistically signifi cant, these probabilities were not reported, because they cannot be interpreted clearly ( 36 ). A one-way ANOVA with Bonferroni's posthoc test with correction for fi ve comparisons (effect of low and high LPS in control and LiCl fed rats, and aCSF effect in LiCl compared with control diet rats) was performed. Data are reported as means (left and right) ± SD with statistical signifi cance set as P р 0.05.

Fatty acid composition of diets
The fatty acid concentrations ( mol/g diet) in the three diets are shown in Table 1 . There was no significant difference among the three diets. The 5001 diet contained (as percent of total fatty acids): 25.2% saturated, 33.3% monounsaturated, 35.1% linoleic, 3.1% ␣ -linolenic, 0.39% AA, 1.25% eicosapentaenoic acid, and 1.62% DHA.

Effect of cannula implantation
Initial experiments investigated the effects, if any, of implanting the cannula and infusing aCSF on brain concentrations of unesterifi ed fatty acids, eicosanoids, and 17-OH-DHA. Except for PGE 2 , the concentration of none of these substances was altered by the cannula implant plus aCSF infusion (data not shown). A very low concentration of PGE 2 (at the limit of detection) was detected in one of four brains of control diet and in one of four brains of lithium diet rats infused with aCSF. These fi ndings show MO; Escherichia coli , serotype 055:B5) at a low dose (1 g/ml at 0.5 ng/h) or a high dose (0.5 mg/ml at 250 ng/h) was infused into the fourth ventricle through the cannula via an osmotic pump (Alzet, Model 2002, Cupertino, CA). Before surgery, the prefi lled pump was placed in sterile 0.9% NaCl at 37°C overnight to start immediate pumping. Postoperative care included triple antibiotic ointment applied to the wound and 5 ml of sterile 0.9% NaCl (sc) to prevent dehydration during recovery. Following 6 days of LPS or aCSF infusion, starting after a rat had been on a control or lithium diet for 36 days, rats were anesthetized with Nembutal ® (40 mg/kg, ip) and subjected to head-focused microwave irradiation (5.5 kW, 3.6 s; Cober Electronics, Stamford, CT). Brains were removed and stored at Ϫ 80°C. In addition, six control and six lithium diet rats, which did not undergo surgery, were anesthetized with Nembutal ® and subjected to head-focused microwave irradiation.

Extraction and analysis of lipids
Brain lipids were extracted with 80% methanol and purifi ed on a C18 column as described previously ( 29 ). Right and left microwaved cerebral hemispheres were homogenized separately in 4 ml of 80% methanol and d 8 -5-HETE, d 8 -AA, d 5 -DHA, d 4 -TXB 2 , d 4 -PGE 2 (Cayman Chemicals, Ann Arbor, MI) as internal standards. Tissue debris was removed by centrifugation and the supernatant was loaded onto a Strata C18-E cartridge (Phenomenex, Torrance, CA). The eluate was taken to dryness and reconstituted in 70 l of HPLC solvent A (8.3 mM acetic acid, pH 5.7) + 20 l of solvent B (acetonitrile-MeOH, 65:35, v/v). A 35 l aliquot of each sample was injected into a HPLC system and subjected to RP-HPLC and eluted at a fl ow rate of 50 l/min, with a linear gradient from 25% to 100% of mobile phase B. Solvent B was increased from 25% to 85% in 24 min, to 100% in 26 min, and held at 100% for a further 12 min. The HPLC effl uent was directly connected to the electrospray source of a triple quadrupole mass spectrometer. Analytes were detected in negative ion mode using multiple reaction monitoring of the specifi c transi-

Brain-specifi c PLA 2 activities
Rats were anesthetized with Nembutal ® and decapitated. Frozen half-hemispheres were homogenized in 3 vols of ice-cold buffer containing 10 mM HEPES, pH 7.5, 1 mM EDTA, 0.34 M sucrose and protease inhibitor cocktail tablet (Roche Diagnostics, Mannheim, Germany). The homogenates were centrifuged at 100,000 g for 1 h at 4°C. Supernatants corresponding to the cytosolic fractions were assayed for cPLA 2 -IV and iPLA 2 -VI activities using the sensitive and specifi c method of Yang et al. ( 35 ) and for sPLA 2 activity using a sPLA 2 assay kit (Cayman Chemical, Ann Arbor, MI).
one-way ANOVAs with Bonferroni posthoc tests showed that high-dose LPS signifi cantly increased brain PGE 2 by 18.5-fold and that the LiCl diet prevented this increase ( Table 2 ). The TXB 2 concentration is not reported, because it was below the limit of detection in each sample. Treatment effects on concentrations of 5-HETE, 5-oxo-ETE, 12-HETE, and 15-HETE also are summarized in Table 2 . A two-way ANOVA showed signifi cant main effects of LiCl on 5-HETE ( P = 0.001) and 5-oxo-ETE ( P < 0.001) and a signifi cant diet × LPS interaction for 15-HETE ( P = 0.02). LiCl increased signifi cantly 5-HETE (mean = 21.70 pmol/g) by 1.8-fold compared with control diet (mean = 12.10 pmol/g; P = 0.0006.). LiCl increased signifi cantly 5-oxo-ETE (mean = 7.64 pmol/g) by 4.3-fold compared with the control diet (mean = 2.36 pmol/g; P < 0.0001). A oneway ANOVA with Bonferroni posthoc tests showed that LiCl increased 15-HETE in high-dose LPS-infused rats but had no signifi cant effect at baseline. Neither the high-nor low-dose LPS had a signifi cant main effect on any of these concentrations.

17-OH-DHA
LC/MS/MS analysis revealed that 17-OH-DHA, monitored by transition m/z 343 → 245, was present in the brain of control diet rats and that its concentration was increased by the LiCl diet ( Fig. 1 ). A two-way ANOVA showed that the LiCl diet had a signifi cant main effect ( P = 0.001) in increasing the concentration of 17-OH-DHA by 1.9-fold (LiCl mean = 0.67 vs. control diet mean = 0.36; P = 0.002) ( Table 2 ). The interaction between LiCl and LPS was insignifi cant, and LPS had no main effect.

PLA 2 activities and 15-LOX-2 protein
A two-way ANOVA on whole brain cPLA 2 -IV and sPLA 2 specifi c activities showed signifi cant diet × LPS interactions, at P = 0.0002 and P < 0.0001, respectively ( Table 3 ). Subsequent one-way ANOVAs with Bonferroni posthoc tests showed that both doses of LPS compared with aCSF signifi cantly increased brain cPLA 2 -IV activity by 36% and a slight occasional effect of cannula implantation, likely due to minimal neuroinfl ammation around the cannula track ( 37 ). In a prior study, PGE 2 could not be detected in control microwaved rat brain in the absence of a cannula ( 29 ). Low-dose LPS-or aCSF-infused rats with indwelling catheters appeared behaviorally normal after 24 h, whereas high-dose LPS-infused rats were lethargic and docile throughout the 6 day infusion period.

Weight and other effects
A two-way ANOVA showed a signifi cant main effect of LPS infusion ( P < 0.0001) but no signifi cant main effect of diet ( P = 0.67) or diet × LPS interaction ( P = 0.32) with regard to body weight (data not shown). A Bonferroni posthoc test indicated that high-dose LPS signifi cantly decreased body weight in both groups by 20% ( P < 0.001), whereas low-dose LPS had a signifi cant effect (7% reduction) only in the control diet rats.

Unesterifi ed fatty acids
Brain concentrations of unesterifi ed AA, DHA, and DPA are summarized in Table 2 . A two-way ANOVA showed a signifi cant diet × LPS interaction for the AA concentration ( P < 0.001). Subsequent one-way ANOVAs with Bonferroni posthoc tests showed that both the low and high doses of LPS compared with aCSF signifi cantly increased brain AA by 31% and 38%, respectively. The LiCl diet prevented the signifi cant increments with both LPS doses. LiCl did not signifi cantly alter the baseline AA concentration (after aCSF infusion). Neither LiCl nor LPS infusion modifi ed DHA or DPA concentrations signifi cantly.

Eicosanoids
A low concentration of PGE 2 at the limit of detection was detected in one of four brains from control as well as from lithium diet rats infused with aCSF. Higher concentrations were found in control diet rats infused with LPS ( Table 2 ). A two-way ANOVA showed a signifi cant diet × LPS interaction for the PGE 2 concentration ( P < 0.001). Subsequent Each value is a mean ± SD, n = 5-6, except for PGE 2 , n = 4. Fatty acids are expressed in nmol/g brain, and eicosanoids and 17-OH-DHA in pmol/g brain.
The mechanism underlying the 17-OH-DHA elevation is uncertain. Because LiCl did not increase the concentration of its precursor, unesterified DHA, or iPLA 2 -VI activity, consistent with prior data ( 20,33 ), the increment may have arisen from enhanced 15-LOX activity. On the other hand, unesterified DHA likely is partitioned in different brain compartments [it is found in neurons and glia ( 41,42 )], as reported for unesterified AA ( 23 ), one of which may be the precursor to 17-OH-DHA. Increased 15-LOX activity is suggested by the increased 15-HETE in the rats fed the LiCl diet during high LPS exposure, because 15-HETE is generated from AA by the action of 15-LOX. Although cytosolic 15-LOX-2 protein level was not significantly increased in the LPS-infused rats fed LiCl, we cannot rule out posttranslational upregulation of 15-LOX activity, which has been reported ( 43 ). Increasing the number of animals in future experiments and measuring membrane 15-LOX protein and activity might be helpful. Whether 15-LOX or other yet-to-be 148%, respectively, and brain sPLA 2 activity by 41% and 80%, respectively. The LiCl diet prevented the signifi cant increment of cPLA 2 -IV activity following low-but not highdose LPS, as well as the signifi cant increments in sPLA 2 activity caused by low-and high-dose LPS. Neither the LiCl diet nor LPS infusion signifi cantly affected whole brain iPLA 2 -VI activity.

DISCUSSION
The major new fi nding of our study is that LiCl increased 17-OH-DHA formation in rat brain with aCSF and LPS infusion. 17-OH-DHA has been reported to have antiinfl ammatory actions. For example, 17-OH-DHA inhibited tumor necrosis factor-␣ (TNF-␣ )-induced interleukin-1 ␤ gene expression in human microglial cells ( 10 ), human neutrophil 5-LOX ( 38 ), and TNF-␣ release and 5-LOX protein expression in murine macrophages ( 39 ). 17-OH-DHA also is an agonist of the tran-  Each value is a mean ± SD, n = 4. Specifi c PLA 2 activities are expressed in pmol/mg protein/min. Data were compared using two-way ANOVA. When LiCl × LPS interactions were signifi cant, a one-way ANOVA with Bonferroni's posttest with correction for fi ve comparisons was performed. * P < 0.05 and *** P < 0.001. centration, consistent with lithium not changing baseline cPLA 2 -IV and sPLA 2 activities. The absence of a LiCl effect on sPLA 2 agrees with a previous report ( 50 ), whereas cP-LA 2 -IV mRNA and protein were downregulated by LiCl in another study ( 51 ). Intravenous or intraperitoneal LPS in rodents has been reported to increase brain sPLA 2 -IIA and sPLA 2 -IIE mRNA, respectively ( 52,53 ). These data suggest that lithium acts differently in a "normal" unstressed brain compared with an "infl ammatory" brain. Lithium might modulate cPLA 2 -IV and sPLA 2 upregulation in response to LPS by decreasing the intracellular Ca 2+ released by glutamate acting at N-methyl-D-aspartic acid receptors (Ca 2+ mediates translocation or phosphorylation of cPLA 2 ) or by reducing the level of phosphatidylinositol 4,5-bisphosphate, which anchors cPLA 2 to perinuclear and nuclear membranes ( 54 ).
This study also investigated possible effects of cannula implantation followed by a 6 day aCSF infusion. Except for a change in PGE 2 , the procedure did not affect any measurement, consistent with the reported little or absence of an infl ammatory reaction under the experimental conditions ( 37 ). Body weight was reduced signifi cantly by LPS infusion, more so by the high than the low dose. Weight loss has been noted with high-dose intracerebroventricular LPS ( 21,22 ) and with peripheral LPS injection ( 55 ). The proinfl ammatory cytokines TNF-␣ , interleukin-1 ␤ , and interleukin-6 have been suggested to play a role in weight loss ( 55 ). Peripheral LPS produces sleepiness and inactivity ( 56 ), both of which were more evident in the high-dose LPS-infused rats.
In summary ( Fig. 2 ), fi nding that LiCl prefeeding upregulated the brain concentration of 17-OH-DHA provides a new possible mechanism for lithium's reported neuroprotective action ( 57 ), in addition to downregulating the AA cascade ( 25,58 ). Supporting such a mechanism is epidemiological evidence that aspirin, which can increase identifi ed enzymes or pathways ( 16 ) are involved in 17-OH-DHA formation following lithium remains to be elucidated.
The LiCl diet increased brain 5-HETE and 5-oxo-ETE without affecting 12-HETE, whereas neither low-nor highdose LPS affected these metabolites. One possible explanation for this observation is that lithium affects AA remodeling within phospholipids by reducing AA-CoA formation ( 33 ) or lysophospholipid acyl CoA transferase activity, making more unesterifi ed AA available to the LOX pathways. Similarly, aspirin, ibuprofen, indomethacin, and valproate, which inhibit cyclooxygenase (COX) activity like lithium, have been reported to increase brain HETE concentrations ( 20,(44)(45)(46)(47).
In this study, high-dose LPS infusion increased brain AA and PGE 2 concentrations and cPLA 2 -IV and sPLA 2 activities without changing the brain DHA concentration or iPLA 2 -VI activity, consistent with evidence that iP-LA 2 -VI is selective for DHA hydrolysis from phospholipid ( 27 ). Although the high-dose LPS signifi cantly increased both cPLA 2 -IV and sPLA 2 activities more than did the low dose, we did not observe a dose-dependent response to LPS in the brain unesterifi ed AA concentration. These data suggest that AA, released by cPLA 2 and sPLA 2 during high-dose LPS infusion, was converted rapidly to eicosanoids and/or reincorporated into brain phospholipids ( 48,49 ). The LiCl diet prevented only the effect on sPLA 2 activity.
The results from this study are consistent with our ischemia study and other reports showing that concentrations of unesterifi ed AA, 17-OH-DHA, 5-and 12-HETEs, and 5-oxo-HETE, measured by RP-HPLC/MS/MS, are much lower in high-energy microwaved than nonmicrowaved brain ( 29,30,32 ). PGE 2 was detected in only one of four brains from control diet rats infused with aCSF. In our previous study using another rodent diet, we could not detect PGE 2 in control microwaved brain (without a cannula) ( 29 ). Additionally, we showed that intracerebrally injected d 4 -PGE 2 was not degraded substantially by the microwaving procedure ( 29 ). These observations indicate that little endogenous PGE 2 is produced in the absence of a brain insult and that the PGE 2 that we could detect in the two brains in this study likely was associated with cannularelated damage ( 37 ). In contrast to our earlier report regarding ischemia ( 29 ), we did not detect E 2 /D 2 isoprostanes in any sample. TXB 2 was reported to be at the limit of detection in microwaved brain ( 29 ), as was the case in the present study.
This study showing that low-dose LPS compared with aCSF infusion in control diet rats signifi cantly increased brain concentrations of AA and PGE 2 but not of DHA, as well as cPLA 2 -IV activity, and that lithium attenuated these changes, confi rms data obtained with different methods ( 23,25 ). In this study, we confi rmed an increased brain sPLA 2 activity by LPS infusion ( 23 ). Dampening by lithium of elevated AA concentrations caused by low-or high-dose LPS is consistent with lithium also dampening the LPSinduced increases in cPLA 2 and sPLA 2 activities. LiCl did not signifi cantly alter the baseline brain unesterifi ed AA con- Fig. 2. LPS infusion increases brain concentration of unesterifi ed AA via cPLA 2 and sPLA 2 and PGE 2 via COX without altering DHA release via iPLA 2 , and LiCl blocks these increases. In addition, LiCl increases levels of 15-HETE, 17-OH-DHA, 5-HETE, and 5-oxo-ETE in the brain of rats subjected to neuroinfl ammation. 17(R)-OH-DHA by acetylating COX-2 ( 9, 59 ), when given chronically reduced untoward effects in (presumably) bipolar disorder patients on lithium therapy ( 60 ). Neuroinfl ammation also has been associated with an upregulated AA cascade in bipolar disorder ( 6,7 ). Lithium's ability to suppress this cascade while stimulating 17-OH-DHA formation may contribute to its effi cacy in bipolar disorder and other neuroinfl ammatory diseases ( 4,5 ). Effi cacy of lithium treatment in HIV-1 dementia ( 61 ), amyotrophic lateral sclerosis ( 62 ), and Alzheimer's disease ( 63 ) has been noted in recent limited clinical trials.