Mol Neurobiol DOI 10.1007/s12035-017-0693-1 Cognitive Impairment and Brain and Peripheral Alterations in a Murine Model of Intraventricular Hemorrhage in the Preterm Newborn Antonio Segado-Arenas1 & Carmen Infante-Garcia2,3 & Isabel Benavente-Fernandez1 & Daniel Sanchez-Sotano1,2 & Juan Jose Ramos-Rodriguez2,3 & Almudena Alonso-Ojembarrena1 & Simon Lubian-Lopez1,4 & Monica Garcia-Alloza2 Received: 9 March 2017 /Accepted: 14 July 2017 # Springer Science+Business Media, LLC 2017 Abstract Germinal matrix hemorrhage-intraventricular hem- newborns, reduced gelsolin levels and increased ubiquitin orrhage (GMH-IVH) remains a serious complication in the carboxy-terminal hydrolase L1 and tau levels were detected preterm newborn. The significant increase of survival rates in GMH-IVH patients at birth. A similar profile was observed in extremelye preterm newborns has also contributed to in- in our mouse model after hemorrhage. Interestingly, early crease the absolute number of patients developing GMH- changes in gelsolin and carboxy-terminal hydrolase L1 levels IVH. However, there are relatively few available animal significantly correlated with the hemorrhage grade in new- models to understand the underlying mechanisms and periph- borns. Altogether, our data support the utility of this animal eral markers or prognostic tools. In order to further character- model to reproduce the central complications and peripheral ize central complications and evolution of GMH-IVH, we changes observed in the clinic, and support the consideration injected collagenase intraventricularly to P7 CD1 mice and of gelsolin, carboxy-terminal hydrolase L1, and tau as feasible assessed them in the short (P14) and the long term (P70). biomarkers to predict the development of GMH-IVH. Early complications at P14 included ventricle enlargement, increased bleeding, and inflammation. These alterations were Keywords Germinal matrix hemorrhage-intraventricular maintained at P70, when increased tau phosphorylation and hemorrhage . Neurogenesis . Tau . Gelsolin . decreased neurogenesis were also observed, resulting in im- Carboxy-terminal hydrolase L1 paired learning and memory in these early adult mice. We additionally analyzed peripheral blood biomarkers in both our mouse model and preterm newborns with GMH-IVH. While MMP9 levels were not significantly altered in mice or Introduction Germinal matrix-intraventricular hemorrhage (GMH-IVH) re- mains a serious problem in the preterm newborn (PTNB). * Simon Lubian-Lopez [email protected] Despite the improvement of neonatal critical care in the last decades, the absolute number of infants who develop severe * Monica Garcia-Alloza [email protected] GMH-IVH has not declined, mostly due to a significant in- crease of the survival rates of the very and extremely preterm 1 Division of Neonatology, Hospital Universitario Puerta del Mar, infant (for review [1]). GMH-IVH results from bleeding from Cadiz, Spain the germinal matrix, a complex vascularized layer also corre- 2 Division of Physiology, School of Medicine, Instituto de sponds with the subventricular zone (SVZ), from where neu- Investigación Biomédica e Innovación en Ciencias Biomédicas de la rons and glial cells rise during fetal development [2]. Provincia de Cádiz (INiBICA), Universidad de Cadiz, Plaza Fragela Germinal matrix starts to involute by 28 weeks of gestational sn, 4 piso 410, Cadiz, Spain age, and it is generally absent in term newborns, while the 3 Salus Infirmorum, Universidad de Cadiz, Cadiz, Spain PTNB are susceptible to local hemorrhage due to the extreme 4 Division of Pediatrics, Hospital Universitario Puerta del Mar, Ava. friability of these capillaries, combined with their inability to Ana de Villa sn, Cadiz, Spain autoregulate cerebral blood flow [2]. While GMH-IVH can Mol Neurobiol occur a few days later, it has been reported that ~80% of all indicates the power of such an animal model and allows for cases occur within the first 72 h after birth. GMH-IVH can future exploration of serum UCH-L1, gelsolin, or tau as pos- expand in the following day, leading to adjacent parenchymal sible predictors of GMH-IVH severity in PTNB patients. involvement and the development of ventriculomegaly [3]. The short- and long-term neurodevelopmental outcomes in infants with severe GMH-IVH are a major concern, as GMH- Material and Methods IVH increases the risk of cerebral palsy and neurodevelopmental or psychomotor alterations [4, 5]. Even Animals and Treatments mild GMH-IVH has been associated with major neurological abnormalities and neurodevelopmental impairment in preterm Male and female CD1 offspring underwent a lesion by a col- infants [6, 7]. Whereas the relevance of these consequences is lagenase VII (Col) (Sigma, St. Louis, MO) intracerebroven- out of question, there is no consensus regarding timing of tricular (icv) infusion at P7, as previously described with mi- intervention, markers, predictors, or treatment [8]. Therefore, nor modifications [11]. Briefly, mice were anesthetized with controllable and reproducible animal models of GMH-IVH isoflurane and immobilized in a stereotactic frame (David- are essential to study feasible markers, therapies, and preven- Kopf, CA, USA); 0.1 or 0.3 IU of Col in 1 μlofTESCA tive measures [9]. In this regard, diverse options have been was administered in the right ventricle (AP −3mm,ML explored with variable success, including physiologic manip- −1 mm, and DV +4 mm, using bregma as a reference) at ulations (hyperosmolar or hypertensive agents, induction of 0.2 μl/min, using a Hamilton syringe, which was left in the hypoxia, hypercapnia, or hypervolemia) or injections of donor lesion site for five more minutes after completing the admin- or autologous blood into the periventricular region (for review istration process. Sham animals underwent the same surgical [10]). On the other hand, intraventricular administration of approach but received 1 μl PBS. Newborn mice were allowed collagenase might provide a simple and reproducible method to recover and returned to the cage with their mothers. All to induce GMH-IVH, as previously shown in rats [9]. animals were weighted before the lesions and before sacrifice, Collagenases are proteolytic intracellular enzymes that hydro- at P14 or P70. For proliferation and neurogenesis studies, lyze collagen, a major component of the basal lamina of blood animals received ip 5-bromo-3-deoxyuridine (BrdU; 70 mg/ vessels. Therefore, direct injection of collagenase dissolves kg) for three consecutive days before sacrifice. All experimen- the extracellular matrix surrounding capillaries, opens the tal procedures were approved by Junta de Andalucia blood-brain barrier, and induces intracranial bleeding [9]. To (Guidelines for Care and Use of Experimental Animals, our knowledge, this approach has not been used in mice, and European Commission Directive 2010/63/UE and Spanish long-term (up to early adulthood) morphopathological and Royal Decree 53/2013) and the University of Cadiz behavioral changes have not been addressed. Moreover, fea- Bioethical Committee. sible markers have not been explored, and observations have not been correlated to outcomes in PTNB patients with GMH- Rotarod IVH. We have found that induction of GMH-IVH to newborn mice results in early central bleeding, brain atrophy, neuronal Motor skills were evaluated in the rotarod (Ugo Basile Srl; loss, impaired neurogenesis, and inflammation. These alter- Varese, Italia) as previously described with minor modifica- ations, as well as increased tau hyperphosphorylation as a tions [12]. Animals were placed on the apparatus for 3 min at marker of neuronal damage, and behavioral impairments are 4 rpm for habituation purposes. During the test probe, the observed up to the early adulthood in collagenase-treated speed was increased from 4 to 60 rpm within 1 min, and the mice. Exploring peripheral feasible markers of GMH-IVH time spent on the rotarod was recorded. complications revealed that while matrix-metalloproteinase 9 (MMP9) was not altered, ubiquitin carboxy-terminal hydro- New Object Discrimination Task lase L1 (UCH-L1), gelsolin, and tau levels were signficantly affected early on in our animal model. Moreover, PTNB with Seven days prior to sacrifice at P70, episodic memory was GMH-IVH exhibited a similar profile of changes in UCH-L1, assessed as previously described [13]. For the purpose of gelsolin, and tau levels at birth. Interestingly, early changes in habituation, mice were exposed to two objects in a trans- UCH-L1 and gelsolin were significantly correlated with hem- parent rectangular box (22 cm long × 44 cm width × 40 cm orrhage severity grade in PTNB patients. Altogether, our high), which were not used again during the object explo- mouse model mimics morphopathological and functional ration task. The next day, each mouse underwent two changes observed in GMH-IVH patients in the short and the sample trials and a test trial. On the first sample trial, long term, allowing for therapeutic alternatives in a chronic mice were placed in the center of a box containing four neuropathological situation to be developed. Additionally, are copies of a novel object (blue balls) arranged in a triangle- similar, in both our animal model and human samples, shaped spatial configuration and were allowed to explore Mol Neurobiol them for 5 min. After a delay of 30 min, the mice Cresyl Violet Staining underwent a second sample trial with four novel objects (red cones), arranged in a quadratic-shaped spatial config-