Human mesenchymal stem cell (MSC) therapy has shown neuroprotective effects and improvement on recovery from neonatal intraventricular hemorrhage (IVH). This study focused on histological evaluations of human amniotic fluid MSC therapy during early prenatal life in a preterm IVH rabbit model. IVH was diagnosed at 24 h of age with ultrasound and animals were randomized into subgroups with confirmed IVH and an IVH negative control group. Animals with confirmed IVH received vehicle only or MSCs at two different doses via intraperitoneal administration. The animals were sacrificed at 48 h post administration. The severity of IVH was histologically analyzed via staining of endogenous peroxidase activity in cryosections and the distribution of red blood cells and cell-free hemoglobin was scored. Primary antibodies targeting human epitopes were validated in IHC assays of frozen MSC pellet. An anti-human nuclear mitotic apparatus (hNuMA) antibody labeled the majority of cells in the MSC pellet and did not cross-react with rabbit NuMA when tested in the nontreated rabbit brain. Significant levels of red blood cells and cell-free hemoglobin were found in the IVH confirmed group, whereas the control group showed no hemorrhage. The MSC therapy groups showed similar scoring results as the IVH-vehicle group. Anti-hNuMA immunolabeling did not detect any cells in the brain of MSC treated rabbits. However, extracellular (nonnuclear) immunolabeling was detected, located in the midbrain of the animals that received MSCs, indicating the presence of MSC nuclear debris. The preterm rabbit model was proven successful for inducing IVH, whereas MSC treatment did not affect the degree of hemorrhage. To increase the possibility to detect the MSCs post administration, future studies should include prelabeling of the MSCs with a suitable cell tracker and analyses at time points closer to the administration of MSCs. / Human mesenkymal stamcellsterapi har visat neuroprotektiva effekter samt förbättring av återhämtning efter neonatal intraventrikulär blödning (IVH). Denna studie fokuserade på histologiska utvärderingar av stamcellsterapi med humana mesenkymala stamceller (MSC) från fostervatten under tidigt prenatalt liv i en prematur IVH-kaninmodell. IVH diagnostiserades med ultraljud vid 24 tim. ålder och djuren delades slumpmässigt in i undergrupper med konfirmerad IVH och en IVH-negativ kontrollgrupp. Djur med konfirmerad IVH mottog endast bärmedel eller MSC i två olika doser via intraperitoneal administration. Djuren avlivades 48 tim. post administration. Graden av IVH analyserades histologiskt genom färgning av peroxidasaktivitet i kryosnitt av hjärna och distributionen av erytrocyter samt fritt hemoglobin graderades. Primärantikroppar riktade mot humana epitop validerades i immunhistokemiska analyser av fryst MSC-pellet. En anti-human nukleär mitotisk apparat (hNuMA) antikropp märkte majoriteten av cellerna i MSC-pelleten och korsreagerade inte med kanin-NuMA under försök i obehandlad kaninhjärna. Signifikanta nivåer av erytrocyter och fritt hemoglobin påvisades i gruppen med konfirmerad IVH, medan kontrollgruppen inte visade någon blödning. Samtliga IVH-grupper; IVH-vehicle och de som mottog MSC- terapi, visade liknande blödningsgrad. Anti-hNuMA-immuninmärkning kunde inte detektera några celler i de MSC-behandlade kaninhjärnorna. Dock detekterades extracellulär (icke-nukleär) immuninmärkning lokaliserad i mitthjärnan hos de djur som mottog MSC, vilket indikerar närvaro av nukleär MSC-debris. Den prematura kaninmodellen bevisades vara framgångsrik för induktion av IVH, men MSC-terapi påverkade inte blödningsgraden. För att öka sannolikheten att detektera MSC efter administration, borde framtida studier inkludera förmärkning av MSC med en lämplig cellmarkör samt analyser vid tidpunkter närmare administrationen av MSC.

Made available in DSpace on 2016-08-19T18:16:08Z (GMT). No. of bitstreams: 1 Roxana Desterro.pdf: 688994 bytes, checksum: ceb8107f09c4ea13f75ca8e4a721da06 (MD5) Previous issue date: 2007-12-13 / The scientific and technologic advances that occurred in the neonatal ITU over the last decades increased the survival rate of babies over and over more premature. Due to the occurency of possible sequelae inherent to this condition, it has been a bigger interest for de development of egress babies from these unites of treatment. The present study is retrospective, longitudinal, analytical of a control case, nested to a cohort. It proposes to evaluate the motor and cognitive development of premature babies who had brain ultrasound alterations at the neonatal period and the possible risk factors for its delay. It has been selected 99 premature children weighting 1800 grams or less in the birth and pregnancy age inferior to 37 weeks, submitted to a transfontanelar ultrasound in the neonatal period during their neonatal ITU (Intensive Therapy Unity) internment. The socio-economic, cultural, environmental, perinatal clinic events and mother characteristics were analysed. To evaluation of the neural, psychomotor development, the Denver II test was used. A sample made, in the majority, of corrected 12 months old children. The birth weight average was 1032 grams and the pregnancy average was 31,3 weeks. The ultrasonic alterations were present in 49, 4% of the children. In them, the periventricular leucomalacy was more frequent corrected one year old babies with alterations in the Denver II test. 34, 3% of the realized tests had unsatisfactory results. As risk factors for the development alteration, Ultrasonic alterations and low family incomes were significant for the study. . The positive predictive value of transfontanelar ultrasonic exams for the neural psychomotor development was 51,02% and the negative predictive value was 82% When the family incomes variable is added to the transfontanelar ultrasonic alterations, the positive predictive value increased to 90% and the negative predictive value decreased to 71,91%. It is believed that the variable family incomes added to the analysis is a good alternative to increase the prediction capacity development alterations of premature children with transfontanelar ultrasonic alterations. / Os avanços científicos e tecnológicos que ocorreram nas UTI neonatais nas últimas décadas, aumentaram a sobrevida de bebês cada vez mais prematuros. Devido ocorrência de possíveis seqüelas inerentes a essa condição, houve um interesse maior pelo desenvolvimento dos bebês egressos dessas unidades de tratamento. O presente estudo é retrospectivo, longitudinal, analítico do tipo caso controle, aninhado a uma coorte. Foi avaliado o desenvolvimento motor e cognitivo de prematuros que tiveram alterações ultra-sonográficas cerebrais no período neonatal e os possíveis fatores de risco para o seu atraso. Selecionou-se 99 crianças prematuras com peso de nascimento menor ou igual a 1800 gramas e idade gestacional abaixo de 37 semanas e que fizeram ultra-sonografia transfontanelar no período neonatal durante sua internação em UTI neonatal. Analisaram-se variáveis sócio-econômicas, culturais, ambientais, eventos clínicos perinatais e características maternas. Para avaliação do desenvolvimento neuropsicomotor utilizou-se o Teste de Denver II. A população foi composta de crianças com 12 meses de idade corrigida. A média de peso de nascimento foi de 1032 gramas e a média de idade gestacional foi de 31,3 semanas. As alterações ultra-sonográficas estiveram presentes em 49,4% das crianças. Destas, a leucomalácia periventricular foi a mais presente nos bebês com alteração no Teste de Denver II na idade corrigida de 1 ano. Dentre os testes realizados, 34,3% tiveram resultados desfavoráveis. Dos fatores de risco para alteração de desenvolvimento, alterações ultra-sonográficas cerebrais e renda familiar mostram-se estatisticamente significantes para o estudo. O valor preditivo positivo dos exames ultra-sonográficos transfontanelares para alterações de desenvolvimento neuropsicomotor, foi de 51,02% e valor preditivo negativo de 82%. Ao se acrescentar a variável renda familiar às alterações ultra-sonográficas transfontanelares, o valor preditivo positivo aumentou para 90% e o valor preditivo negativo reduziu-se para 71,91%. Acredita-se que o acréscimo à análise da variável renda familiar baixa é boa alternativa para aumentar a capacidade de predição de alterações do desenvolvimento de prematuros com alterações ultra-sonográficas transfontanelares.

Abstract Children born preterm are prone to acute brain insults related to subsequent neurodevelopmental impairments. However, the role of specific biomarkers and perinatal clinical factors in the pathogenesis of brain injury and neurodevelopmental sequelae has remained poorly understood. The present study evaluated whether specific immunoproteins at birth predict the risk of intraventricular hemorrhage (IVH) and whether their receptors are localized at the bleeding site. We further investigated whether children who went on to develop cerebral palsy (CP) could be identified on the basis of blood immunoproteins collected during the perinatal period. The association between single nucleotide polymorphisms in the chemokine CCL18 gene and susceptibility to CP was also studied. Finally, we investigated the association of pre- and postnatal factors with cognitive outcomes in very preterm-born schoolchildren without impairments. The present study revealed that a low concentration of CCL18 in cord blood was an independent risk factor of IVH in very preterm infants. The CCL18 receptor, CCR3, was detectable in the periventricular area and in the neurons of the hippocampus in preterm infants already at 23 weeks of gestation. We also identified a cluster of cord blood cytokines that was associated with the risk of CP. In addition, inflammatory cytokine levels were associated with CP risk on days 1 and 7 after birth. The genetic study showed that both IVH and the CCL18 polymorphism independently and additively had an influence on CP susceptibility. Our study further demonstrated that schoolchildren born very preterm without CP or cognitive impairment had poorer performance in visuospatial–sensorimotor skills and in attention–executive functions than term-born children. Fetal growth restriction was an independent risk factor of compromised neurocognitive outcome in very preterm children predicting difficulties in language, memory and learning. In conclusion, specific cytokines and cytokine clusters serve as biomarkers of different pathways involved in damage to the brain structures and in the pathogenesis of CP. In addition, genetic factors can affect these processes. Further, fetal growth restriction and prematurity play important roles in neurocognitive development later in life. / Tiivistelmä Hyvin ennenaikaisina syntyneet lapset ovat alttiita akuuteille aivovaurioille sekä myöhemmin ilmeneville kehityshäiriöille. Eri välittäjäaineiden sekä raskaudenaikaisten ja syntymänjälkeisten kliinisten tekijöiden vaikutusta aivojen vaurioherkkyyteen sekä neurologiseen ja neurokognitiiviseen kehitykseen ei kuitenkaan ole tutkittu riittävästi. Tässä tutkimuksessa tarkasteltiin, ennustaako jokin napaverestä tutkituista sytokiineista aivoverenvuotoa hyvin ennenaikaisesti syntyneillä vastasyntyneillä. Lisäksi selvitettiin, onko sytokiinin spesifinen reseptori osoitettavissa vuotoherkällä alueella aivoissa. Tutkimme myös, ennustaako jokin napaveren immunoproteiini-profiilin komponentti CP-vamman syntyä joko itsenäisesti tai yhdessä muiden perinataalisten riskitekijöiden kanssa sekä lisääkö tietyn sytokiinin (CCL18) geneettinen vaihtelu CP-vamman riskiä hyvin ennenaikaisesti syntyneillä lapsilla. Lisäksi selvitimme, vaikuttavatko raskaudenaikaiset tekijät ja vastasyntyneisyyskauden sairaudet neurokognitiiviseen kehitykseen kouluiässä. Tämän tutkimuksen mukaan napaveren matala CCL18-kemokiinipitoisuus oli itsenäinen aivoverenvuodon riskitekijä. CCR3-reseptori, johon CCL18 sitoutuu, oli osoitettavissa sekä vuotoherkällä alueella että hermosoluissa 23. raskausviikon iästä lähtien. Havaitsimme myös, että tietyt napaveren sytokiiniryppäät ja yksittäisten tulehdusvastevälittäjäaineiden pitoisuudet 1. ja 7. elinpäivänä olivat yhteydessä CP-riskiin. Lisäksi havaitsimme yhteyden CCL18-kemokiinin geneettisen vaihtelun ja aivoverenvuodon sekä CP-vamman kehittymisen välillä. Tutkimuksemme mukaan hyvin ennenaikaisesti syntyneet koululaiset, joilla ei ollut CP- tai kehitysvammaa, suoriutuivat täysiaikaisina syntyneitä verrokkeja heikommin visuaalista hahmotusta ja sensomotoriikkaa sekä tarkkaavuutta ja toiminnanohjausta mittaavissa testeissä. Lisäksi havaitsimme sikiöaikaisen kasvuhäiriön ennustavan itsenäisesti heikkoa suoritusta kieltä, muistia ja oppimista testaavissa tehtävissä ennenaikaisesti syntyneillä lapsilla. Tietyt sytokiinit ja sytokiiniryppäät ovat yhteydessä aivovauriomekanismeihin. Nämä mekanismit saattavat yhdessä perinnöllisen alttiuden kanssa vaikuttaa myös CP-vamman syntyyn. Sikiöaikainen kasvuhäiriö ja ennenaikaisuus vaikuttavat lapsen myöhempään neurokognitiiviseen kehitykseen.