This study aims to determine whether sickle cell mice could recapitulate features of cognitive and neurobehavioral impairment observed in sickle cell patients and whether neuroinflammation could be a potential therapeutic target as in other non-sickle cell disease-related cognitive dysfunction. Cognitive (learning and memory) and behavioral (anxiety) deficits in 13- and later 6-month-old male Townes humanized sickle cell (SS) and matched control (AA) mice were evaluated using novel object recognition (NOR) and fear conditioning tests. Immunohistochemistry was performed to quantify peripheral immune cell (CD45+) and activated microglia (Iba1+) as markers of neuroinflammation in the dentate and peri-dentate gyrus areas. We evaluated cell fate by measuring 5'-bromodeoxyuridine and doublecortin fluorescence and phenotyped proliferating cells using either glial fibrillary acid protein (GFAP+), neuronal nuclei (NeuN+), CD45+, and Iba1+. In addition, Golgi-Cox staining was used to assess markers of neuroplasticity (dendritic spine density and morphology and density of dendrite arbors) on cortical and hippocampal pyramidal neurons. Compared to matched AA controls, 13-month-old SS mice showed significant evidence of cognitive and behavioral deficit on NOR and fear conditioning tests. Also, SS mice had significantly higher density of CD45+ and activated microglia cells (i.e. more evidence of neuroinflammation) in the dentate and peri-dentate gyrus area. Additionally, SS mice had significantly lower dendritic spine density, but a higher proportion of immature dendritic spines. Treatment of 13-month-old SS mice with minocycline resulted in improvement of cognitive and behavioral deficit compared to matched vehicle-treated SS mice. Also, treated SS mice had significantly fewer CD45+ and activated microglia cells (i.e. less evidence of neuroinflammation) in the dentate and peri-dentate gyrus, as well as a significant improvement in markers of neuroplasticity.
Impact statement
This study provides crucial information that could be helpful in the development of new or repurposing of existing therapies for the treatment of cognitive deficit in individuals with sickle cell disease (SCD). Its impact is in demonstrating for the first time that neuroinflammation and along with abnormal neuroplasticity are among the underlying mechanism of cognitive and behavioral deficits in SCD and that drugs such as minocycline which targets these pathophysiological mechanisms could be repurposed for the treatment of this life altering complication of SCD.
Ohene-FrempongKWeinerSJSleeperLAMillerSTEmburySMoohrJWWethersDLPegelowCHGillFM.Sickle cell disease tCSo. Cerebrovascular accidents in sickle cell disease: rates and risk factors. Blood1998;
91:288–94
2.
AndreottiCKingAAMacyECompasBEDeBaunMR.The association of cytokine levels with cognitive function in children with sickle cell disease and normal MRI studies of the brain. J Child Neurol2015;
30:1349–53
3.
BentonTDBoydRIfeagwuJFeldtmoseESmith-WhitleyK.Psychiatric diagnosis in adolescents with sickle cell disease: a preliminary report. Curr Psychiatry Rep2011;
13:111–5
4.
BentonTDIfeagwuJASmith-WhitleyK.Anxiety and depression in children and adolescents with sickle cell disease. Curr Psychiatry Rep2007;
9:114–21
5.
MahdiNAl-OlaKKhalekNAAlmawiWY.Depression, anxiety, and stress comorbidities in sickle cell anemia patients with vaso-occlusive crisis. J Pediatr Hematol/Oncol2010;
32:345–9
6.
AnieKA.Psychological complications in sickle cell disease. Br J Haematol2005;
129:723–9
7.
DeWaltDAGrossHEGipsonDSSelewskiDTDeWittEMDampierCDHindsPSHuangICThissenDVarniJW.PROMIS((R)) pediatric self-report scales distinguish subgroups of children within and across six common pediatric chronic health conditions. Qual Life Res2015;
24:2195–208
8.
RouthieauxJSarconeSStegengaK.Neurocognitive sequelae of sickle cell disease: current issues and future directions. J Pediatr Oncol Nurs2005;
22:160–7
9.
De MontalembertMWangW.Cerebrovascular complications in children with sickle cell disease. Handb Clin Neurol2013;
113:1937–43
10.
KingAADeBaunMRWhiteDA.Need for cognitive rehabilitation for children with sickle cell disease and strokes. Expert Rev Neurother2008;
8:291–6
11.
ArmstrongFDThompsonRJJr.WangWZimmermanRPegelowCHMillerSMoserFBelloJHurtigAVassK. Cognitive functioning and brain magnetic resonance imaging in children with sickle cell disease. Neuropsychology committee of the cooperative study of sickle cell disease. Pediatrics1996;
97:864–70
12.
KingAAStrouseJJRodeghierMJCompasBECasellaJFMcKinstryRCNoetzelMJQuinnCTIchordRDowlingMMMillerJPDebaunMR.Parent education and biologic factors influence on cognition in sickle cell anemia. Am J Hematol2014;
89:162–7
13.
SchatzJWhiteDAMoinuddinAArmstrongMDeBaunMR.Lesion burden and cognitive morbidity in children with sickle cell disease. J Child Neurol2002;
17:891–5
14.
VichinskyEPNeumayrLDGoldJIWeinerMWRuleRRTruranDKastenJEgglestonBKeslerKMcMahonLOrringerEPHarringtonTKalinyakKDe CastroLMKutlarARutherfordCJJohnsonCBessmanJDJordanLBArmstrongFD.The Neuropsychological Dysfunction Neuroimaging Adult Sickle Cell Anemia Study Group. Neuropsychological dysfunction and neuroimaging abnormalities in neurologically intact adults with sickle cell anemia. JAMA2010;
303:1823–31
15.
WoodDH.Cerebrovascular complications of sickle cell anemia. Stroke1978;
9:735
16.
Al-JafarHAAlroughaniRAAbdullahTAl-QallafF.Neurological complications in sickle cell disease. Int J Clin Exp Neurol2016;
4:9–18
17.
PrenglerMPavlakisSGProhovnikIAdamsRJ.Sickle cell disease: the neurological complications. Ann Neurol2002;
51:543–52
18.
VenkataramanAAdamsRJ.Neurologic complications of sickle cell disease. Handb Clin Neurol2014;
120:1015–25
19.
McKimDBNiraulaATarrAJWohlebESSheridanJFGodboutJP.Neuroinflammatory dynamics underlie memory impairments after repeated social defeat. J Neurosci2016;
36:2590–604
QinLWuXBlockMLLiuYBreeseGRHongJSKnappDJCrewsFT.Systemic LPS causes chronic neuroinflammation and progressive neurodegeneration. Glia2007;
55:453–62
22.
WohlebESPowellNDGodboutJPSheridanJF.Stress-induced recruitment of bone marrow-derived monocytes to the brain promotes anxiety-like behavior. J Neurosci2013;
33:13820–33
23.
BrownCEBoydJDMurphyTH.Longitudinal in vivo imaging reveals balanced and branch-specific remodeling of mature cortical pyramidal dendritic arbors after stroke. J Cereb Blood Flow Metab2010;
30:783–91
24.
BrownCELiPBoydJDDelaneyKRMurphyTH.Extensive turnover of dendritic spines and vascular remodeling in cortical tissues recovering from stroke. J Neurosci2007;
27:410109
25.
TakumaKHaraYKataokaSKawanaiTMaedaYWatanabeRTakanoEHayata-TakanoAHashimotoHAgoYMatsudaT.Chronic treatment with valproic acid or sodium butyrate attenuates novel object recognition deficits and hippocampal dendritic spine loss in a mouse model of autism. Pharmacol Biochem Behav2014;
126:43–9
26.
BedrosianTAFonkenLKWaltonJCHaimANelsonRJ.Dim light at night provokes depression-like behaviors and reduces CA1 dendritic spine density in female hamsters. Psychoneuroendocrinology2011;
36:1062–9
27.
ClementJPAcetiMCresonTKOzkanEDShiYReishNJAlmonteAGMillerBHWiltgenBJMillerCAXuXRumbaughG.Pathogenic SYNGAP1 mutations impair cognitive development by disrupting maturation of dendritic spine synapses. Cell2012;
151:709–23
28.
KnutsonDCMitzeyAMTaltonLEClagett-DameM.Mice null for NEDD9 (HEF1) display extensive hippocampal dendritic spine loss and cognitive impairment. Brain Res2016;
1632:141–55
29.
LevensonJLMcClishDKDahmanBABovbjergVEdeACVPenberthyLTAisikuIPRobertsJDRoseffSDSmithWR.Depression and anxiety in adults with sickle cell disease: the PiSCES project. Psychosom Med2008;
70:192–6
30.
SogutluALevensonJLMcClishDKRosefSDSmithWR.Somatic symptom burden in adults with sickle cell disease predicts pain, depression, anxiety, health care utilization, and quality of life: the PiSCES project. Psychosomatics2011;
52:272–9
31.
National Research council. Guide for the care and use of laboratory animals.
Washington, DC:
National Academies Press, 2010.
32.
BramhallMFlorez-VargasOStevensRBrassACruickshankS.Quality of methods reporting in animal models of colitis. Inf lamm Bowel Dis2015;
21:1248–59
33.
WangLAlmeidaLEde Souza BatistaCMKhaibullinaAXuNAlbaniSGuthKASeoJSQuezadoMQuezadoZM.Cognitive and behavior deficits in sickle cell mice are associated with profound neuropathologic changes in hippocampus and cerebellum. Neurobiol Dis2016;
85:60–72
34.
HyacinthHISugiharaCLSpencerTLArcherDRShihAY.Higher prevalence of spontaneous cerebral vasculopathy and cerebral infarcts in a mouse model of sickle cell disease. J Cereb Blood Flow Metab2019;
39:342–51
35.
HyacinthHICapersPLArcherDRHibbertJM.TNF-α, IFN-γ, IL-10, and IL-4 levels were elevated in a murine model of human sickle cell anemia maintained on a high protein/calorie diet. Exp Biol Med2014;
239:65–70
36.
ManciEAHyacinthHICapersPLArcherDRPittsSGhoshSPatricksonJTitfordMEOfori-AcquahSFHibbertJM.High protein diet attenuates histopathologic organ damage and vascular leakage in transgenic murine model of sickle cell anemia. Exp Biol Med2014;
239:966–74
37.
WohlebESFennAMPacentaAMPowellNDSheridanJFGodboutJP.Peripheral innate immune challenge exaggerated microglia activation, increased the number of inflammatory CNS macrophages, and prolonged social withdrawal in socially defeated mice. Psychoneuroendocrinology2012;
37:1491–505
38.
ColovicMCacciaS.Liquid chromatographic determination of minocycline in brain-to-plasma distribution studies in the rat. J Chromatogr B Analyt Technol Biomed Life Sci2003;
791:337–43
39.
ChoiYKimHSShinKYKimEMKimMKimHSParkCHJeongYHYooJLeeJPChangKAKimSSuhYH.Minocycline attenuates neuronal cell death and improves cognitive impairment in Alzheimer's disease models. Neuropsychopharmacology2007;
32:2393–404
40.
GarcezMLMinaFBellettini-SantosTCarneiroFGLuzAPSchiavoGLAndrighettiMSScheidMGBolfeRPBudniJ.Minocycline reduces inflammatory parameters in the brain structures and serum and reverses memory impairment caused by the administration of amyloid beta (1–42) in mice. Prog Neuropsychopharmacol Biol Psychiatry2017;
77:23–31
41.
SharmaRKimS-YSharmaAZhangZKambhampatiSPKannanSKannanRM.Activated microglia targeting dendrimer-minocycline conjugate as therapeutics for neuroinflammation. Bioconjug Chem2017;
28:2874–86
42.
BilousovaTVDansieLNgoMAyeJCharlesJREthellDWEthellIM.Minocycline promotes dendritic spine maturation and improves behavioural performance in the fragile X mouse model. J Med Genet2009;
46:94–102
43.
TaoTFengJZXuGHFuJLiXGQinXY.Minocycline promotes neurite outgrowth of PC12 cells exposed to oxygen-glucose deprivation and reoxygenation through regulation of MLCP/MLC signaling pathways. Cell Mol Neurobiol2017;
37:417–26
44.
PaxinosGFranklinKB.The mouse brain in stereotaxic coordinates.
Houston, TX:
Gulf Professional Publishing, 2004.
45.
DonnellyDJGenselJCAnkenyDPvan RooijenNPopovichPG.An efficient and reproducible method for quantifying macrophages in different experimental models of central nervous system pathology. J Neurosci Methods2009;
181:36–44
46.
ZhaoCTengEMSummersRGMingG-LGageFH.Distinct morphological stages of dentate granule neuron maturation in the adult mouse hippocampus. J Neurosci2006;
26:3–11
47.
BowmanRELuineVDiaz WeinsteinSKhandakerHDeWolfSFrankfurtM.Bisphenol-A exposure during adolescence leads to enduring alterations in cognition and dendritic spine density in adult male and female rats. Horm Behav2015;
69:89–97
48.
GlasperERLaMarcaEABocarslyMEFasolinoMOpendakMGouldE.Sexual experience enhances cognitive flexibility and dendritic spine density in the medial prefrontal cortex. Neurobiol Learn Mem2015;
125:73–9
49.
YuHWangDDWangYLiuTLeeFSChenZY.Variant brain-derived neurotrophic factor Val66Met polymorphism alters vulnerability to stress and response to antidepressants. J Neurosci2012;
32:4092–101
50.
HarrisKM.Structure, development, and plasticity of dendritic spines. Curr Opin Neurobiol1999;
9:343–8
51.
BermanRFMurrayKDArqueGHunsakerMRWenzelHJ.Abnormal dendrite and spine morphology in primary visual cortex in the CGG knock-in mouse model of the fragile X premutation. Epilepsia2012;
53:150–60
52.
AndolinaDConversiDCabibSTrabalzaAVenturaRPuglisi-AllegraSPascucciT.5-Hydroxytryptophan during critical postnatal period improves cognitive performances and promotes dendritic spine maturation in genetic mouse model of phenylketonuria. Int J Neuropsychopharmacol2011;
14:479–89
53.
StankoJPEasterlingMRFentonSE.Application of Sholl analysis to quantify changes in growth and development in rat mammary gland whole mounts. Reprod Toxicol2015;
54:129–35
54.
ShollDA.Dendritic organization in the neurons of the visual and motor cortices of the cat. J Anat1953;
87:387–406
55.
RistanovicDMilosevicNTStulicV.Application of modified Sholl analysis to neuronal dendritic arborization of the cat spinal cord. J Neurosci Methods2006;
158:212–8
56.
MilosevicNTRistanovicD.The Sholl analysis of neuronal cell images: semi-log or log-log method?J Theor Biol2007;
245:130–40
57.
SpychalaMSHonarpishehPMcCulloughLD.Sex differences in neuroinflammation and neuroprotection in ischemic stroke. J Neurosci Res2017;
95:462–71
58.
ThompsonRJJr.GustafsonKEBonnerMJWareRE.Neurocognitive development of young children with sickle cell disease through three years of age. J Pediatr Psychol2002;
27:235–44
59.
PegelowCHWangWGrangerSHsuLLVichinskyEMoserFGBelloJZimmermanRAAdamsRJBrambillaD,
Trial ftS. Silent infarcts in children with sickle cell anemia and abnormal cerebral artery velocity. Arch Neurol2001;
58:2017–21
60.
BernaudinFVerlhacSArnaudCKamdemAVasileMKasbiFHauIMadhiFFourmauxCBiscardiSEpaudRPondarreC.Chronic and acute anemia and extracranial internal carotid stenosis are risk factors for silent cerebral infarcts in sickle cell anemia. Blood2015;
125:1653–61
61.
DeBaunMRSarnaikSARodeghierMJMinnitiCPHowardTHIyerRVInusaBTelferPTKirby-AllenMQuinnCTBernaudinFAireweleGWoodsGMPanepintoJAFuhBKwiatkowskiJKKingAARhodesMMThompsonAAHeinyMERedding-LallingerRCKirkhamFJSabioHGonzalezCESaccenteSLKalinyakKAStrouseJJFixlerJMGordonMOMillerJPNoetzelMJIchordRNCasellaJF.Associated risk factors for silent cerebral infarcts in sickle cell anemia: low baseline hemoglobin, sex, and relative high systolic blood pressure. Blood2012;
119:3684–90
62.
KwiatkowskiJLZimmermanRAPollockANSetoWSmith-WhitleyKShultsJBlackwood-ChirchirAOhene-FrempongK.Silent infarcts in young children with sickle cell disease. Br J Haematol2009;
146:300–5
63.
BlazerDG.Cognitive neuroscience of aging: linking cognitive and cerebral aging. Am J Psychiatry2006;
163:560–1
KassimAAPruthiSDayMRodeghierMGindvilleMCBrodskyMADeBaunMRJordanLC.Silent cerebral infarcts and cerebral aneurysms are prevalent in adults with sickle cell anemia. Blood2016;
127:2038–40
66.
SangerMJordanLPruthiSDayMCovertBMerriweatherBRodeghierMDeBaunMKassimA.Cognitive deficits are associated with unemployment in adults with sickle cell anemia. J Clin Exp Neuropsychol2016;
38:661–71
67.
SteenRGFineberg-BuchnerCHankinsGWeissLPrifiteraAMulhernRK.Cognitive deficits in children with sickle cell disease. J Child Neurol2005;
20:102–7
68.
RuffieuxNNjamnshiAKWonkamAHauertCAChanalJVerdonVFonsahJYEtaSCDohRFNgamaleuRNKengneAMFossatiCSztajzelR.Association between biological markers of sickle cell disease and cognitive functioning amongst Cameroonian children. Child Neuropsychol2013;
19:143–60
69.
AtakaKAsakawaANagaishiKKaimotoKSawadaAHayakawaYTatezawaRInuiAFujimiyaM.Bone marrow-derived microglia infiltrate into the paraventricular nucleus of chronic psychological stress-loaded mice.PloS One2013;
8:e81744
70.
SawadaANiiyamaYAtakaKNagaishiKYamakageMFujimiyaM.Suppression of bone marrow-derived microglia in the amygdala improves anxiety-like behavior induced by chronic partial sciatic nerve ligation in mice. Pain2014;
155:1762–72
71.
BorosBDGreathouseKMGentryEGCurtisKABirchallELGearingMHerskowitzJH.Dendritic spines provide cognitive resilience against Alzheimer's disease. Ann Neurol2017;
82:602–14
72.
Sa-NguanmooPTanajakPKerdphooSSatjaritanunPWangXLiangGLiXJiangCPratchayasakulWChattipakornNChattipakornSC.FGF21 improves cognition by restored synaptic plasticity, dendritic spine density, brain mitochondrial function and cell apoptosis in obese-insulin resistant male rats. Horm Behav2016;
85:86–95
73.
CaoZYangXZhangHWangHHuangWXuFZhuangCWangXLiY.Aluminum chloride induces neuroinflammation, loss of neuronal dendritic spine and cognition impairment in developing rat. Chemosphere2016;
151:289–95
74.
RiceRASpangenbergEEYamate-MorganHLeeRJAroraRPHernandezMXTennerAJWestBLGreenKN.Elimination of microglia improves functional outcomes following extensive neuronal loss in the hippocampus. J Neurosci2015;
35:9977–89
75.
KohmanRABhattacharyaTKKilbyCBuckoPRhodesJS.Effects of minocycline on spatial learning, hippocampal neurogenesis and microglia in aged and adult mice. Behav Brain Res2013;
242:17–24
76.
JiangYLiuYZhuCMaXMaLZhouLHuangQCenLPiRChenX.Minocycline enhances hippocampal memory, neuroplasticity and synapse-associated proteins in aged C57 BL/6 mice. Neurobiol Learn Mem2015;
121:20–9
77.
MinYLiHXuKHuangYXiaoJWangWLiLYangTHuangLYangLJiangHWangQZhaoMHuaHMeiRLiF.Minocycline-suppression of early peripheral inflammation reduces hypoxia-induced neonatal brain injury. Front Neurosci2017;
11:511
ChavesCMarqueCRTrzesniakCMachado de SousaJPZuardiAWCrippaJADursunSMHallakJE.Glutamate-N-methyl-D-aspartate receptor modulation and minocycline for the treatment of patients with schizophrenia: an update. Braz J Med Biol Res2009;
42:1002–14
83.
DziembowskaMPrettoDIJanuszAKaczmarekLLeighMJGabrielNDurbin-JohnsonBHagermanRJTassoneF.High MMP-9 activity levels in fragile X syndrome are lowered by minocycline. Am J Med Genet A2013;
161a:1897–903
84.
GiriPKLuYLeiSLiWZhengJLuHChenXLiuYZhangP.Pretreatment with minocycline improves neurogenesis and behavior performance after midazolam exposure in neonatal rats. Neuroreport2018;
29:153–9
85.
HanYZhangLWangQZhangDZhaoQZhangJXieLLiuGYouZ.Minocycline inhibits microglial activation and alleviates depressive-like behaviors in male adolescent mice subjected to maternal separation. Psychoneuroendocrinology2019;
107:37–45
86.
FortierLAMottaTGreenwaldRADiversTJMayrKG.Synoviocytes are more sensitive than cartilage to the effects of minocycline and doxycycline on IL-1alpha and MMP-13-induced catabolic gene responses. J Orthop Res2010;
28:522–8
87.
SillerSSBroadieK.Matrix metalloproteinases and minocycline: therapeutic avenues for fragile X syndrome. Neural Plast2012;
2012:124548
88.
ChenSDYinJHHwangCSTangCMYangDI.Anti-apoptotic and anti-oxidative mechanisms of minocycline against sphingomyelinase/ceramide neurotoxicity: implication in Alzheimer's disease and cerebral ischemia. Free Radic Res2012;
46:940–50
89.
TangCMHwangCSChenSDYangDI.Neuroprotective mechanisms of minocycline against sphingomyelinase/ceramide toxicity: roles of bcl-2 and thioredoxin. Free Radic Biol Med2011;
50:710–21
90.
MatteiDDjodari-IraniAHadarRPelzAde CossioLFGoetzTMatyashMKettenmannHWinterCWolfSA.Minocycline rescues decrease in neurogenesis, increase in microglia cytokines and deficits in sensorimotor gating in an animal model of schizophrenia. Brain Behav Immun2014;
38:175–84
91.
VaySUBlaschkeSKleinRFinkGRSchroeterMRuegerMA.Minocycline mitigates the gliogenic effects of proinflammatory cytokines on neural stem cells. J Neurosci Res2016;
94:149–60
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