Neuroprotective Effect of Oligodendrocyte Precursor Cell Transplantation in a Long-Term Model of Periventricular Leukomalacia
2009; Elsevier BV; Volume: 175; Issue: 6 Linguagem: Inglês
10.2353/ajpath.2009.090051
ISSN1525-2191
AutoresDaniel J. Webber, Marka van Blitterswijk, Siddharthan Chandran,
Tópico(s)Infant Development and Preterm Care
ResumoPerinatal white matter injury, or periventricular leukomalacia (PVL), is the most common cause of brain injury in premature infants and is the leading cause of cerebral palsy. Despite increasing numbers of surviving extreme premature infants and associated long-term neurological morbidity, our understanding and treatment of PVL remains incomplete. Inflammation- or ischemia/hypoxia-based rodent models, although immensely valuable, are largely restricted to reproducing short-term features of up to 3 weeks after injury. Given the long-term sequelae of PVL, there is a need for subchronic models that will enable testing of putative neuroprotective therapies. Here, we report long term characterization of a neonatal inflammation-induced rat model of PVL. We show bilateral ventriculomegaly, inflammation, reactive astrogliosis, injury to pre-oligodendrocytes, and neuronal loss 8 weeks after injury. We demonstrate neuroprotective effects of oligodendrocyte precursor cell transplantation. Our findings present a subchronic model of PVL and highlight the tissue protective effects of oligodendrocyte precursor cell transplants that demonstrate the potential of cell-based therapy for PVL. Perinatal white matter injury, or periventricular leukomalacia (PVL), is the most common cause of brain injury in premature infants and is the leading cause of cerebral palsy. Despite increasing numbers of surviving extreme premature infants and associated long-term neurological morbidity, our understanding and treatment of PVL remains incomplete. Inflammation- or ischemia/hypoxia-based rodent models, although immensely valuable, are largely restricted to reproducing short-term features of up to 3 weeks after injury. Given the long-term sequelae of PVL, there is a need for subchronic models that will enable testing of putative neuroprotective therapies. Here, we report long term characterization of a neonatal inflammation-induced rat model of PVL. We show bilateral ventriculomegaly, inflammation, reactive astrogliosis, injury to pre-oligodendrocytes, and neuronal loss 8 weeks after injury. We demonstrate neuroprotective effects of oligodendrocyte precursor cell transplantation. Our findings present a subchronic model of PVL and highlight the tissue protective effects of oligodendrocyte precursor cell transplants that demonstrate the potential of cell-based therapy for PVL. Premature, low-birth weight infants are commonly diagnosed with perinatal white matter damage, which leads to long-term neurological deficits including cerebral palsy.1Back SA Rivkees SA Emerging concepts in periventricular white matter injury.Semin Perinatol. 2004; 28: 405-414Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 2Drougia A Giapros V Krallis N Theocharis P Nikaki A Tzoufi M Andronikou S Incidence and risk factors for cerebral palsy in infants with perinatal problems: a 15-year review.Early Hum Dev. 2007; 83: 541-547Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 3Resch B Vollaard E Maurer U Haas J Rosegger H Muller W Risk factors and determinants of neurodevelopmental outcome in cystic periventricular leucomalacia.Eur J Pediatr. 2000; 159: 663-670Crossref PubMed Scopus (101) Google Scholar, 4Pierrat V Duquennoy C van HI Ernst M Guilley N de Vries LS Ultrasound diagnosis and neurodevelopmental outcome of localised and extensive cystic periventricular leucomalacia.Arch Dis Child Fetal Neonatal Ed. 2001; 84: F151-F156Crossref PubMed Google Scholar Congenital encephalomyelitis was described almost 150 years ago, a disease in newborn children characterized by pale softened zones of degeneration within the deep white matter surrounding lateral ventricles and is now often referred to as periventricular leukomalacia (PVL).5Banker BQ Larroche JC Periventricular leukomalacia of infancy. A form of neonatal anoxic encephalopathy.Arch Neurol. 1962; 7: 386-410Crossref PubMed Scopus (565) Google Scholar The white matter damage that occurs in preterm infants has more recently been shown to be also accompanied by significant cerebral-cortex and deep-gray matter abnormalities leading to neurodegeneration and altered neurobehavioral performance.6Leviton A Gressens P Neuronal damage accompanies perinatal white-matter damage.Trends Neurosci. 2007; 30: 473-478Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar PVL is characterized by selective oligodendrocyte precursor cell (OPC) loss resulting in delayed or disrupted myelination, white matter atrophy and ventriculomegaly, neuronal loss, and cyst and scar formation. Extreme premature birth (approximately 23 to 32 weeks) accompanied by inflammation or infection corresponds to the period when immature dividing and differentiating oligodendrocytes predominate in the cerebral white matter.7Blumenthal I Periventricular leucomalacia: a review.Eur J Pediatr. 2004; 163: 435-442Crossref PubMed Scopus (84) Google Scholar, 8Khwaja O Volpe JJ Pathogenesis of cerebral white matter injury of prematurity.Arch Dis Child Fetal Neonatal Ed. 2008; 93: F153-F161Crossref PubMed Scopus (507) Google Scholar, 9Volpe JJ Neurobiology of periventricular leukomalacia in the premature infant.Pediatr Res. 2001; 50: 553-562Crossref PubMed Scopus (825) Google Scholar, 10Leviton A Gilles F Ventriculomegaly, delayed myelination, white matter hypoplasia, and "periventricular" leukomalacia: how are they related?.Pediatr Neurol. 1996; 15: 127-136Abstract Full Text PDF PubMed Scopus (170) Google Scholar PVL has a complex etiology. The two most important determinants are cerebral hypoperfusion and maternal intrauterine infection. Periventricular white matter is susceptible to hypoperfusion due to the comparative immaturity of the periventricular vasculature of the preterm infant.11Inage YW Itoh M Takashima S Correlation between cerebrovascular maturity and periventricular leukomalacia.Pediatr Neurol. 2000; 22: 204-208Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 12Nakamura Y Okudera T Hashimoto T Vascular architecture in white matter of neonates: its relationship to periventricular leukomalacia.J Neuropathol Exp Neurol. 1994; 53: 582-589Crossref PubMed Scopus (67) Google Scholar Early oligodendrocyte lineage cells are vulnerable to the consequences of hypoperfusion and subsequent microglial and astrocytic activation on account of amplified glutamate receptor-mediated responses and lack of efficient antioxidant protection.13Bernardo A Greco A Levi G Minghetti L Differential lipid peroxidation, Mn superoxide, and bcl-2 expression contribute to the maturation-dependent vulnerability of oligodendrocytes to oxidative stress.J Neuropathol Exp Neurol. 2003; 62: 509-519PubMed Google Scholar, 14Karadottir R Hamilton NB Bakiri Y Attwell D Spiking and nonspiking classes of oligodendrocyte precursor glia in CNS white matter.Nat Neurosci. 2008; 11: 450-456Crossref PubMed Scopus (269) Google Scholar, 15Back SA Han BH Luo NL Chricton CA Xanthoudakis S Tam J Arvin KL Holtzman DM Selective vulnerability of late oligodendrocyte progenitors to hypoxia-ischemia.J Neurosci. 2002; 22: 455-463PubMed Google Scholar, 16Ness JK Romanko MJ Rothstein RP Wood TL Levison SW Perinatal hypoxia-ischemia induces apoptotic and excitotoxic death of periventricular white matter oligodendrocyte progenitors.Dev Neurosci. 2001; 23: 203-208Crossref PubMed Scopus (128) Google Scholar, 17Back SA Gan X Li Y Rosenberg PA Volpe JJ Maturation-dependent vulnerability of oligodendrocytes to oxidative stress-induced death caused by glutathione depletion.J Neurosci. 1998; 18: 6241-6253Crossref PubMed Google Scholar, 18Back SA Riddle A McClure MM Maturation-dependent vulnerability of perinatal white matter in premature birth.Stroke. 2007; 38: 724-730Crossref PubMed Scopus (297) Google Scholar Maternal infection, recently implicated as a causative factor in the pathogenesis of PVL,19Boggess KA Pathophysiology of preterm birth: emerging concepts of maternal infection.Clin Perinatol. 2005; 32: 561-569Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 20Cai Z Pang Y Lin S Rhodes PG Differential roles of tumor necrosis factor-alpha and interleukin-1 beta in lipopolysaccharide-induced brain injury in the neonatal rat.Brain Res. 2003; 975: 37-47Crossref PubMed Scopus (161) Google Scholar, 21Leviton A Paneth N Reuss ML Susser M Allred EN Dammann O Kuban K Van Marter LJ Pagano M Hegyi T Hiatt M Sanocka U Shahrivar F Abiri M Disalvo D Doubilet P Kairam R Kazam E Kirpekar M Rosenfeld D Schonfeld S Share J Collins M Genest D Shen-Schwarz S Developmental Epidemiology Network Investigators. Maternal infection, fetal inflammatory response, and brain damage in very low birth weight infants.Pediatr Res. 1999; 46: 566-575Crossref PubMed Scopus (359) Google Scholar, 22Cai Z Pan ZL Pang Y Evans OB Rhodes PG Cytokine induction in fetal rat brains and brain injury in neonatal rats after maternal lipopolysaccharide administration.Pediatr Res. 2000; 47: 64-72Crossref PubMed Scopus (437) Google Scholar, 23Pang Y Cai Z Rhodes PG Disturbance of oligodendrocyte development, hypomyelination and white matter injury in the neonatal rat brain after intracerebral injection of lipopolysaccharide.Brain Res Dev Brain Res. 2003; 140: 205-214Crossref PubMed Scopus (215) Google Scholar is believed to initiate an inflammatory/cytokine cascade that results in the release of early-response pro-inflammatory cytokines such as tumor necrosis factor–α (TNF-α), interleukin−1 β (IL-1β) and interleukin−6 (IL-6), and causes damage to immature oligodendrocytes.24Kadhim H Tabarki B Verellen G De PC Rona AM Sebire G Inflammatory cytokines in the pathogenesis of periventricular leukomalacia.Neurology. 2001; 56: 1278-1284Crossref PubMed Scopus (313) Google Scholar TNF-α appears to have a particularly important role in PVL pathogenesis with in vitro evidence suggesting direct damage to OPCs,25Andrews T Zhang P Bhat NR TNFalpha potentiates IFNgamma-induced cell death in oligodendrocyte progenitors.J Neurosci Res. 1998; 54: 574-583Crossref PubMed Scopus (144) Google Scholar, 26Cammer W Effects of TNFalpha on immature and mature oligodendrocytes and their progenitors in vitro.Brain Res. 2000; 864: 213-219Crossref PubMed Scopus (82) Google Scholar while IL-1β and IL-6 modulate injury indirectly.27Pang Y Fan LW Zheng B Cai Z Rhodes PG Role of interleukin-6 in lipopolysaccharide-induced brain injury and behavioral dysfunction in neonatal rats.Neuroscience. 2006; 141: 745-755Crossref PubMed Scopus (44) Google Scholar, 28Takahashi JL Giuliani F Power C Imai Y Yong VW Interleukin-1beta promotes oligodendrocyte death through glutamate excitotoxicity.Ann Neurol. 2003; 53: 588-595Crossref PubMed Scopus (214) Google Scholar Although cells of the oligodendrocyte lineage are regarded as the primary target in the pathogenesis of PVL, there is increasing evidence that neonatal white matter damage is accompanied by gray matter abnormalities, including neuronal loss, impaired axonal guidance, and altered synaptogenesis.6Leviton A Gressens P Neuronal damage accompanies perinatal white-matter damage.Trends Neurosci. 2007; 30: 473-478Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 29Pierson CR Folkerth RD Billiards SS Trachtenberg FL Drinkwater ME Volpe JJ Kinney HC Gray matter injury associated with periventricular leukomalacia in the premature infant.Acta Neuropathol. 2007; 114: 619-631Crossref PubMed Scopus (258) Google Scholar Premature newborns affected by PVL often have smaller cerebral cortex and deep gray matter volumes, reduced cortical neurons, and alterations in the orientation of central white matter fiber tracts.6Leviton A Gressens P Neuronal damage accompanies perinatal white-matter damage.Trends Neurosci. 2007; 30: 473-478Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 30Inder TE Warfield SK Wang H Huppi PS Volpe JJ Abnormal cerebral structure is present at term in premature infants.Pediatrics. 2005; 115: 286-294Crossref PubMed Scopus (727) Google Scholar, 31Inder TE Wells SJ Mogridge NB Spencer C Volpe JJ Defining the nature of the cerebral abnormalities in the premature infant: a qualitative magnetic resonance imaging study.J Pediatr. 2003; 143: 171-179Abstract Full Text Full Text PDF PubMed Scopus (439) Google Scholar, 32Peterson BS Vohr B Staib LH Cannistraci CJ Dolberg A Schneider KC Katz KH Westerveld M Sparrow S Anderson AW Duncan CC Makuch RW Gore JC Ment LR Regional brain volume abnormalities and long-term cognitive outcome in preterm infants.JAMA. 2000; 284: 1939-1947Crossref PubMed Scopus (808) Google Scholar Together, these data suggest that developing neurons, like immature oligodendrocyte lineage cells, are also vulnerable to injury caused by inflammatory response or hypoxia. Aspects of PVL have been modeled by ischemia/hypoxia and inflammation-mediated rodent models. Animal models of hypoxia-ischemia have clearly shown that following brain injury there is reduced myelination, enlarged ventricles, loss of neurons and damage to axons and dendrites and altered neurobehavioral performances.33Vannucci SJ Hagberg H Hypoxia-ischemia in the immature brain.J Exp Biol. 2004; 207: 3149-3154Crossref PubMed Scopus (371) Google Scholar, 34Vannucci RC Connor JR Mauger DT Palmer C Smith MB Towfighi J Vannucci SJ Rat model of perinatal hypoxic-ischemic brain damage.J Neurosci Res. 1999; 55: 158-163Crossref PubMed Scopus (302) Google Scholar, 35Follett PL Deng W Dai W Talos DM Massillon LJ Rosenberg PA Volpe JJ Jensen FE Glutamate receptor-mediated oligodendrocyte toxicity in periventricular leukomalacia: a protective role for topiramate.J Neurosci. 2004; 24: 4412-4420Crossref PubMed Scopus (287) Google Scholar, 36Cai Z Lin S Fan LW Pang Y Rhodes PG Minocycline alleviates hypoxic-ischemic injury to developing oligodendrocytes in the neonatal rat brain.Neuroscience. 2006; 137: 425-435Crossref PubMed Scopus (128) Google Scholar, 37Fan LW Lin S Pang Y Rhodes PG Cai Z Minocycline attenuates hypoxia-ischemia-induced neurological dysfunction and brain injury in the juvenile rat.Eur J Neurosci. 2006; 24: 341-350Crossref PubMed Scopus (88) Google Scholar, 38Wang X Rousset CI Hagberg H Mallard C Lipopolysaccharide-induced inflammation and perinatal brain injury.Semin Fetal Neonatal Med. 2006; 11: 343-353Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar, 39Hagberg H Peebles D Mallard C Models of white matter injury: comparison of infectious, hypoxic-ischemic, and excitotoxic insults.Ment Retard Dev Disabil Res Rev. 2002; 8: 30-38Crossref PubMed Scopus (369) Google Scholar Experimentally induced inflammation has been used in a number of studies to model PVL pathology and test potential treatments.23Pang Y Cai Z Rhodes PG Disturbance of oligodendrocyte development, hypomyelination and white matter injury in the neonatal rat brain after intracerebral injection of lipopolysaccharide.Brain Res Dev Brain Res. 2003; 140: 205-214Crossref PubMed Scopus (215) Google Scholar, 40Fan LW Pang Y Lin S Rhodes PG Cai Z Minocycline attenuates lipopolysaccharide-induced white matter injury in the neonatal rat brain.Neuroscience. 2005; 133: 159-168Crossref PubMed Scopus (114) Google Scholar, 41Cai Z Lin S Rhodes PG Neuroprotective effects of N-acetylaspartylglutamate in a neonatal rat model of hypoxia-ischemia.Eur J Pharmacol. 2002; 437: 139-145Crossref PubMed Scopus (41) Google Scholar, 42Fan LW Tien LT Mitchell HJ Rhodes PG Cai Z Alpha-phenyl-n-tert-butyl-nitrone ameliorates hippocampal injury and improves learning and memory in juvenile rats following neonatal exposure to lipopolysaccharide.Eur J Neurosci. 2008; 27: 1475-1484Crossref PubMed Scopus (48) Google Scholar Administration of the endotoxin lipopolysaccharide (LPS), a potent inducer of innate immune response and inflammation,43Fitzgerald KA Rowe DC Golenbock DT Endotoxin recognition and signal transduction by the TLR4/MD2-complex.Microbes Infect. 2004; 6: 1361-1367Crossref PubMed Scopus (224) Google Scholar, 44Lehnardt S Lachance C Patrizi S Lefebvre S Follett PL Jensen FE Rosenberg PA Volpe JJ Vartanian T The toll-like receptor TLR4 is necessary for lipopolysaccharide-induced oligodendrocyte injury in the CNS.J Neurosci. 2002; 22: 2478-2486PubMed Google Scholar either intracerebrally during early neonatal period, intrauterine or peritoneally to a pregnant mother results in inflammation and hypomyelination.20Cai Z Pang Y Lin S Rhodes PG Differential roles of tumor necrosis factor-alpha and interleukin-1 beta in lipopolysaccharide-induced brain injury in the neonatal rat.Brain Res. 2003; 975: 37-47Crossref PubMed Scopus (161) Google Scholar, 22Cai Z Pan ZL Pang Y Evans OB Rhodes PG Cytokine induction in fetal rat brains and brain injury in neonatal rats after maternal lipopolysaccharide administration.Pediatr Res. 2000; 47: 64-72Crossref PubMed Scopus (437) Google Scholar, 23Pang Y Cai Z Rhodes PG Disturbance of oligodendrocyte development, hypomyelination and white matter injury in the neonatal rat brain after intracerebral injection of lipopolysaccharide.Brain Res Dev Brain Res. 2003; 140: 205-214Crossref PubMed Scopus (215) Google Scholar, 38Wang X Rousset CI Hagberg H Mallard C Lipopolysaccharide-induced inflammation and perinatal brain injury.Semin Fetal Neonatal Med. 2006; 11: 343-353Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar Moreover, in animal models of LPS-induced PVL neuronal loss and a reduction in neurite length in the parietal cortex has been observed.42Fan LW Tien LT Mitchell HJ Rhodes PG Cai Z Alpha-phenyl-n-tert-butyl-nitrone ameliorates hippocampal injury and improves learning and memory in juvenile rats following neonatal exposure to lipopolysaccharide.Eur J Neurosci. 2008; 27: 1475-1484Crossref PubMed Scopus (48) Google Scholar, 45Lehnardt S Massillon L Follett P Jensen FE Ratan R Rosenberg PA Volpe JJ Vartanian T Activation of innate immunity in the CNS triggers neurodegeneration through a Toll-like receptor 4-dependent pathway.Proc Natl Acad Sci USA. 2003; 100: 8514-8519Crossref PubMed Scopus (852) Google ScholarIn vitro evidence suggests LPS is not directly toxic to OPCs, but causes injury through the activation of Toll-like receptor 4-positive microglia, which is a source of pro-inflammatory cytokines, nitric oxide, and free radicals.44Lehnardt S Lachance C Patrizi S Lefebvre S Follett PL Jensen FE Rosenberg PA Volpe JJ Vartanian T The toll-like receptor TLR4 is necessary for lipopolysaccharide-induced oligodendrocyte injury in the CNS.J Neurosci. 2002; 22: 2478-2486PubMed Google Scholar, 45Lehnardt S Massillon L Follett P Jensen FE Ratan R Rosenberg PA Volpe JJ Vartanian T Activation of innate immunity in the CNS triggers neurodegeneration through a Toll-like receptor 4-dependent pathway.Proc Natl Acad Sci USA. 2003; 100: 8514-8519Crossref PubMed Scopus (852) Google Scholar, 46Pang Y Cai Z Rhodes PG Effects of lipopolysaccharide on oligodendrocyte progenitor cells are mediated by astrocytes and microglia.J Neurosci Res. 2000; 62: 510-520Crossref PubMed Scopus (83) Google Scholar Furthermore, it has been shown that LPS-induced inflammation increases the susceptibility of white matter to injury in response to otherwise "harmless" subthreshold hypoxic-ischemic insult.47Eklind S Mallard C Leverin AL Gilland E Blomgren K Mattsby-Baltzer I Hagberg H Bacterial endotoxin sensitizes the immature brain to hypoxic–ischaemic injury.Eur J Neurosci. 2001; 13: 1101-1106Crossref PubMed Google Scholar Current models of PVL are typically short term, with studies using either hypoxia-ischemia or inflammation rarely extending analysis beyond 14 days. There is therefore a need to evaluate the longer-term, subchronic consequences of LPS injury and specifically address whether hypomyelination and neuronal injury are self-limiting or indeed spontaneously repair. Cellular therapeutic strategies are predicated on cell replacement and/or tissue protection independent of specific cellular differentiation. Progenitor cells including OPCs have previously been used for cellular replacement of damaged or lost cells in a variety of CNS injury models where damage to myelin and neuronal loss occur.48Lindvall O Kokaia Z Stem cells for the treatment of neurological disorders.Nature. 2006; 441: 1094-1096Crossref PubMed Scopus (685) Google Scholar, 49Foote AK Blakemore WF Inflammation stimulates remyelination in areas of chronic demyelination.Brain. 2005; 128: 528-539Crossref PubMed Scopus (189) Google Scholar, 50Bambakidis NC Miller RH Transplantation of oligodendrocyte precursors and sonic hedgehog results in improved function and white matter sparing in the spinal cords of adult rats after contusion.Spine J. 2004; 4: 16-26Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 51Sharp J Keirstead HS Therapeutic applications of oligodendrocyte precursors derived from human embryonic stem cells.Curr Opin Biotechnol. 2007; 18: 434-440Crossref PubMed Scopus (43) Google Scholar, 52Zhao C Zawadzka M Roulois AJ Bruce CC Franklin RJ Promoting remyelination in multiple sclerosis by endogenous adult neural stem/precursor cells: defining cellular targets.J Neurol Sci. 2008; 265: 12-16Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 53Windrem MS Schanz SJ Guo M Tian GF Washco V Stanwood N Rasband M Roy NS Nedergaard M Havton LA Wang S Goldman SA Neonatal chimerization with human glial progenitor cells can both remyelinate and rescue the otherwise lethally hypomyelinated shiverer mouse.Cell Stem Cell. 2008; 2: 553-565Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar Furthermore, there is accumulating evidence that implicates progenitor cell mediated neuroprotection through a variety of mechanisms including graft derived neurotrophic support independent of directed differentiation.54Martino G Pluchino S The therapeutic potential of neural stem cells.Nat Rev Neurosci. 2006; 7: 395-406Crossref PubMed Scopus (641) Google Scholar, 55Pluchino S Furlan R Martino G Cell-based remyelinating therapies in multiple sclerosis: evidence from experimental studies.Curr Opin Neurol. 2004; 17: 247-255Crossref PubMed Scopus (59) Google Scholar Moreover, there is evidence to suggest that OPCs secrete factors that are capable of supporting neuronal survival,56Wilkins A Compston A Trophic factors attenuate nitric oxide mediated neuronal and axonal injury in vitro: roles and interactions of mitogen-activated protein kinase signalling pathways.J Neurochem. 2005; 92: 1487-1496Crossref PubMed Scopus (42) Google Scholar, 57Wilkins A Majed H Layfield R Compston A Chandran S Oligodendrocytes promote neuronal survival and axonal length by distinct intracellular mechanisms: a novel role for oligodendrocyte-derived glial cell line-derived neurotrophic factor.J Neurosci. 2003; 23: 4967-4974PubMed Google Scholar, 58Wilkins A Chandran S Compston A A role for oligodendrocyte-derived IGF-1 in trophic support of cortical neurons.Glia. 2001; 36: 48-57Crossref PubMed Scopus (150) Google Scholar, 59Anderson TJ Schneider A Barrie JA Klugmann M McCulloch MC Kirkham D Kyriakides E Nave KA Griffiths IR Late-onset neurodegeneration in mice with increased dosage of the proteolipid protein gene A role for oligodendrocyte-derived IGF-1 in trophic support of cortical neurons.J Comp Neurol. 1998; 394: 506-519Crossref PubMed Scopus (118) Google Scholar, 60Griffiths I Klugmann M Anderson T Yool D Thomson C Schwab MH Schneider A Zimmermann F McCulloch M Nadon N Nave KA Axonal swellings and degeneration in mice lacking the major proteolipid of myelin.Science. 1998; 280: 1610-1613Crossref PubMed Scopus (710) Google Scholar, 61Lappe-Siefke C Goebbels S Gravel M Nicksch E Lee J Braun PE Griffiths IR Nave KA Disruption of Cnp1 uncouples oligodendroglial functions in axonal support and myelination.Nat Genet. 2003; 33: 366-374Crossref PubMed Scopus (798) Google Scholar thus suggesting they may be a potential therapeutic source for replacing lost or damaged cells and protecting healthy tissue following neonatal brain injury. In this study we have examined the subchronic effects of LPS induced injury and then examined the putative neuroprotective effects of OPC transplantation. Intracerebral injections of LPS (1 mg/kg, n = 25) or saline (n = 14) to 5-day-old (P5) rats were performed as previously described.23Pang Y Cai Z Rhodes PG Disturbance of oligodendrocyte development, hypomyelination and white matter injury in the neonatal rat brain after intracerebral injection of lipopolysaccharide.Brain Res Dev Brain Res. 2003; 140: 205-214Crossref PubMed Scopus (215) Google Scholar Briefly, postnatal day 5 (P5) Sprague Dawley rats (Harlan, UK) were anesthetized by placing them on ice, before being placed in full body mold to prevent movement during the injection. Bregma was identified using white light lamp. A needle was then used to puncture the skin and skull at the following coordinates from bregma: anteroposterior (AP) −1 mm, medial-lateral (ML) −1 mm, dorsal-ventral (DV) −2 mm.22Cai Z Pan ZL Pang Y Evans OB Rhodes PG Cytokine induction in fetal rat brains and brain injury in neonatal rats after maternal lipopolysaccharide administration.Pediatr Res. 2000; 47: 64-72Crossref PubMed Scopus (437) Google Scholar The dose of LPS was chosen based on the results of previous studies demonstrating white and gray matter injury.22Cai Z Pan ZL Pang Y Evans OB Rhodes PG Cytokine induction in fetal rat brains and brain injury in neonatal rats after maternal lipopolysaccharide administration.Pediatr Res. 2000; 47: 64-72Crossref PubMed Scopus (437) Google Scholar, 23Pang Y Cai Z Rhodes PG Disturbance of oligodendrocyte development, hypomyelination and white matter injury in the neonatal rat brain after intracerebral injection of lipopolysaccharide.Brain Res Dev Brain Res. 2003; 140: 205-214Crossref PubMed Scopus (215) Google Scholar Using a 10-μl precise Hamilton micropipette, a 1-μl solution of LPS (10 mg/kg) was infused over 3 minutes into the periventricular area of the neonatal rat brain (see Supplemental Figure 1 at http://ajp.amjpathol.org). The needle was left in situ for a further 3 minutes before slowly being withdrawn. The P5 rats were then placed on a heated blanket before being return to the mother. On weaning the rats were given food and water ad libitum. All cell culture reagents were obtained from Invitrogen/Gibco (UK) unless otherwise specified. Experimental animals in this study were used under the United Kingdom Animals (Scientific Procedures) Act 1986. Green fluorescent protein (GFP) neonatal rats [gift from Dr. Okabe, postnatal day (P)0-2] were terminated by lethal injection of Euthatal (sodium pentobarbital, 0.05 ml). Following removal of meninges, cortex was isolated and manually minced before enzymatic digestion with 0.1% trypsin/EDTA, followed by 0.001% DNase (Sigma, UK) and centrifugation at 1500 rpm for 5 minutes. The resulting cell pellet was resuspended in triturating solution (containing 0.05% w/v trypsin inhibitor, 1% w/v bovine serum albumin, and 0.002% w/v DNase in Hanks balanced salt solution) and triturated using a flamed glass pipette to obtain a single cell suspension. To obtain OPC populations for transplantation neonatal cerebral cortex suspensions were seeded onto flasks (Poly-D-Lysine-coated, approximately 1.5 × 106 cells seeded/T75 flasks) and maintained in serum buffer [Dulbecco's modified Eagle's medium (4.5 g/L glucose) supplemented with 1% penicillin-streptomycin-fungizone, and 10% fetal calf serum, v/v] for 7 days with complete media changes 24 hours, 3 days, and 6 days, as previously described.62Webber DJ Compston A Chandran S Minimally manipulated oligodendrocyte precursor cells retain exclusive commitment to the oligodendrocyte lineage following transplantation into intact and injured hippocampus.Eur J Neurosci. 2007; 26: 1791-1800Crossref PubMed Scopus (4) Google Scholar One day before transplantation experiments, GFP-mixed glial preparations (7 days) were shaken in an incubated chamber for 1 hour to remove top dwelling microglia. Media was removed and fresh Dulbecco's modified Eagle's medium (with 1% penicillin-streptomycin-fungizone, 10% fetal calf serum) was then added and the mixed glial preparations returned to incubator for at least 1 hour. Flasks were then shaken overnight (16 to 18 hours) to remove the OPCs ready for transplantation. OPCs were maintained as a single cell suspension at 8.0 × 104 cells/μl in Hanks balanced salt solution on ice. The care and treatment of the animals was in accordance with United Kingdom Animals (Scientific Procedures) Act 1986. For the transplantation experiments, Sprague-Dawley postnatal day 11 (P11) neonatal animals previously lesioned at P5 were anesthetized using isoflurane (see Supplemental Figure 1 at http://ajp.amjpathol.org). GFP-OPCs were implanted 1 week after injury. Animals were divided into three groups: LPS-lesioned followed by GFP-OPC transplant (n = 8); LPS-lesioned, followed by vehicle control transplant (n = 8); and control-lesioned, no transplant (saline, n = 6). Animals were placed into a neonatal adaptor to prevent movement during the injection. Coordinates were taken from bregma: AP −1 mm and ML −1 mm. GFP-OPCs (8.0 × 104 cells/μl) or saline control was then drawn into a Hamilton syringe and 1 μl injected unilaterally above the ventricle at a depth of DV −2 mm over 3 minutes. The syringe was left in situ for a further 3 minutes before withdrawing slowly. The pup was then placed on a heated blanket before being returned to the mother. Animal groups (n = 6) were terminated at 8 weeks (when the rats reached young adulthood, see Supplemental Figure 1 at http://ajp.amjpathol.org) following LPS lesioning at P5, using an overdose of sodium pentobarbital (80 mg/kg i.p.), and perfused transcardially with 0.1 M/L PBS followed by 4% paraformaldehyde in 0.1 M/L PBS (pH 7.4). The brains were then
Referência(s)