A Model of Perinatal Ischemic Stroke in the Rat: 20 Years Already and What Lessons?

Neonatal hypoxia-ischemia (HI) and ischemia are a common cause of neonatal brain injury resulting in cerebral palsy with subsequent learning disabilities and epilepsy. Recent data suggest a higher incidence of focal ischemia-reperfusion located in the middle cerebral artery (MCA) territory in near-term and newborn babies. Pre-clinical studies in the field of cerebral palsy research used, and still today, the classical HI model in the P7 rat originally described by Rice et al. (1). At the end of the 90s, we designed a new model of focal ischemia in the P7 rat to explore the short and long-term pathophysiology of neonatal arterial ischemic stroke, particularly the phenomenon of reperfusion injury and its sequelae (reported in 1998). Cerebral blood-flow and cell death/damage correlates have been fully characterized. Pharmacologic manipulations have been applied to the model to test therapeutic targets. The model has proven useful for the study of seizure occurrence, a clinical hallmark for neonatal ischemia in babies. Main pre-clinical findings obtained within these 20 last years are discussed associated to clinical pattern of neonatal brain damage

Inflammatory responses in the cerebral cortex after ischemia in the P7 neonatal Rat.

International audienceBACKGROUND AND PURPOSE: The contribution of inflammatory response to the pathogenesis of ischemic lesions in the neonate is still uncertain. This study described the chronological sequence of inflammatory changes that follow cerebral ischemia with reperfusion in the neonatal P7 rat. METHODS: P7 rats underwent left middle cerebral artery electrocoagulation associated with 1-hour left common carotid artery occlusion. The spatiotemporal pattern of cellular responses was characterized immunocytochemically with the use of antibodies against rat endogenous immunoglobulins to visualize the area of the breakdown of the blood-brain barrier. Infiltration of neutrophils and T lymphocytes was demonstrated by antibodies against myeloperoxidase and a pan-T cell marker, respectively. Antibodies ED1 and OX-42 were applied to identify microglial cells and macrophages. The response of astrocytes was shown with antibodies against glial fibrillary acidic protein. Cell survival was assessed by Bcl-2 expression. Cell death was demonstrated by DNA fragmentation with the use of the terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end labeling (TUNEL) assay and Bax immunodetection. RESULTS: Endogenous immunoglobulin extravasation through the blood-brain barrier occurred at 2 hours of recirculation and persisted until 1 month after ischemia. Neutrophil infiltration began at 24 hours and peaked at 72 to 96 hours (30+/-3.4 neutrophils per 0.3 mm(2); P<0.0001), then disappeared at 14 days after ischemia. T cells were observed between 24 and 96 hours of reperfusion. Resident microglia-macrophages exhibited morphological remnants and expressed the cell death inhibitor Bcl-2 at 24 hours of recirculation. They became numerous within the next 48 hours and peaked at 7 days after ischemia. Phenotypic changes of resident astrocytes were apparent at 24 hours, and they proliferated between 48 hours and 7 days after ischemia. Progressively inflammatory cells showed DNA fragmentation and the cell death activator Bax expression. Cell elimination continued until there was a complete disappearance of the frontoparietal cortex. CONCLUSIONS: These data demonstrate that perinatal ischemia with reperfusion triggers acute inflammatory responses with granulocytic cell infiltration, which may be involved in accelerating the destructive processes

Glial activation in white matter following ischemia in the neonatal P7 rat brain

This study examines cell death and proliferation in the white matter after neonatal stroke. In post-natal day 7 injured rat, there was a marked reduction in myelin basic protein (MBP) immunostaining mainly corresponding to numerous pyknotic immature oligodendrocytes and TUNEL-positive astrocytes in the ipsilateral external capsule. In contrast, a substantial restoration of MBP, as indicated by the MBP ratio of left-toright, occurred in the cingulum at 48 (1.27 +- 0.12) and 72 (1.30 +- 0.18, p<0.05) hours of recovery as compared to age-matched controls (1.03 +- 0.14). Ki-67 immunostaining revealed a first peak of newly-generated cells in the dorsolateral hippocampal subventricular zone and cingulum at 72 hours after reperfusion. Double immunofluorescence revealed that most of the Ki-67-positive cells were astrocytes at 48 hours and NG2 pre-oligodendrocytes at 72 hours of recovery. Microglia infiltration occurs over several days in the cingulum and a huge quantity of macrophages reached the subcortical white matter where they engulfed immature oligodendrocytes. The overall results suggest that the persistent activation of microglia involves a chronic component of immunoinflammation, which overwhelms repair processes and contributes to cystic growth in the developing brain.Comment: 30 page

Activation-Induced Apoptosis of Autoreactive and Alloreactive T Lymphocytes in the Target Organ as a Major Mechanism of Tolerance

Normal individuals have mature T lymphocytes that are capable of reacting to self-antigens and can be activated by cross-reacting environmental antigens. The mechanism that maintains immune tolerance and prevents these activated autoreactive T cells from causing autoimmune disease is unclear. We have previously hypothesized that activation-induced apoptosis of previously activated autoreactive T cells in the target organ is a major mechanism for maintaining tolerance. Here I review the current evidence to support this hypothesis. It is proposed that when activated autoreactive T cells enter the target organ, they are reactivated mainly by non-professional antigen-presenting cells (APC) and deleted by activation-induced apoptosis through the Fas (CD95) pathway before producing significant target organ damage. This apoptosis occurs because the reactivated T cells do not receive sufficient costimulation from the non-professional APC to up-regulate their expression of Bcl-2-related anti-apoptotic proteins, which inhibit the CD95 pro-apoptotic pathway. This is in contrast to the situation in peripheral lymphoid organs, where reactivation of T cells by professional APC results in sufficient costimulation-induced up-regulation of Bcl-2-related proteins to inhibit the CD95 pathway and allow T cell proliferation and survival as memory T cells. Activation-induced apoptosis of alloreactive T cells in allografts can similarly account for spontaneous allograft acceptance, as occurs after MHC-mismatched liver transplantation

Melatonin Promotes Oligodendroglial Maturation of Injured White Matter in Neonatal Rats

OBJECTIVE:To investigate the effects of melatonin treatment in a rat model of white matter damage (WMD) in the developing brain. Additionally, we aim to delineate the cellular mechanisms of melatonin effect on the oligodendroglial cell lineage. METHODS:A unilateral ligation of the uterine artery in pregnant rat at the embryonic day 17 induces fetal hypoxia and subsequent growth restriction (GR) in neonatal pups. GR and control pups received a daily intra-peritoneal injection of melatonin from birth to post-natal day (P) 3. RESULTS:Melatonin administration was associated with a dramatic decrease in microglial activation and astroglial reaction compared to untreated GR pups. At P14, melatonin prevented white matter myelination defects with an increased number of mature oligodendrocytes (APC-immunoreactive) in treated GR pups. Conversely, melatonin was not found to be associated with an increased density of total oligodendrocytes (Olig2-immunoreactive), suggesting that melatonin is able to promote oligodendrocyte maturation but not proliferation. These effects appear to be melatonin-receptor dependent and were reproduced in vitro. INTERPRETATION:These data suggest that melatonin has a strong protective effect on developing damaged white matter through decreased microglial activation and oligodendroglial maturation leading to a normalization of the myelination process. Consequently, melatonin should be a considered as an effective neuroprotective candidate not only in perinatal brain damage but also in inflammatory and demyelinating diseases observed in adults

Neuroprotective Effect of Inhaled Nitric Oxide on Excitotoxic-Induced Brain Damage in Neonatal Rat

BACKGROUND: Inhaled nitric oxide (iNO) is one of the most promising therapies used in neonates. However, little information is known about its impact on the developing brain submitted to excitotoxic challenge. METHODOLOGY/PRINCIPAL FINDINGS: We investigated here the effect of iNO in a neonatal model of excitotoxic brain lesions. Rat pups and their dams were placed in a chamber containing 20 ppm NO during the first week of life. At postnatal day (P)5, rat pups were submitted to intracranial injection of glutamate agonists. At P10, rat pups exposed to iNO exhibited a significant decrease of lesion size in both the white matter and cortical plate compared to controls. Microglia activation and astrogliosis were found significantly decreased in NO-exposed animals. This neuroprotective effect was associated with a significant decrease of several glutamate receptor subunits expression at P5. iNO was associated with an early (P1) downregulation of pCREB/pAkt expression and induced an increase in pAkt protein concentration in response to excitotoxic challenge (P7). CONCLUSION: This study is the first describe and investigate the neuroprotective effect of iNO in neonatal excitotoxic-induced brain damage. This effect may be mediated through CREB pathway and subsequent modulation of glutamate receptor subunits expression

Preparation of Pre-Confluent Retinal Cells Increases Graft Viability In Vitro and In Vivo: A Mouse Model

PURPOSE: Graft failure remains an obstacle to experimental subretinal cell transplantation. A key step is preparing a viable graft, as high levels of necrosis and apoptosis increase the risk of graft failure. Retinal grafts are commonly harvested from cell cultures. We termed the graft preparation procedure "transplant conditions" (TC). We hypothesized that culture conditions influenced graft viability, and investigated whether viability decreased following TC using a mouse retinal pigment epithelial (RPE) cell line, DH01. METHODS: Cell viability was assessed by trypan blue exclusion. Levels of apoptosis and necrosis in vitro were determined by flow cytometry for annexin V and propidium iodide and Western blot analysis for the pro- and cleaved forms of caspases 3 and 7. Graft viability in vivo was established by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and cleaved caspase 3 immunolabeling of subretinal allografts. RESULTS: Pre-confluent cultures had significantly less nonviable cells than post-confluent cultures (6.6%±0.8% vs. 13.1%±0.9%, p<0.01). Cell viability in either group was not altered significantly following TC. Caspases 3 and 7 were not altered by levels of confluence or following TC. Pre-confluent cultures had low levels of apoptosis/necrosis (5.6%±1.1%) that did not increase following TC (4.8%±0.5%). However, culturing beyond confluence led to progressively increasing levels of apoptosis and necrosis (up to 16.5%±0.9%). Allografts prepared from post-confluent cultures had significantly more TUNEL-positive cells 3 hours post-operatively than grafts of pre-confluent cells (12.7%±3.1% vs. 4.5%±1.4%, p<0.001). Subretinal grafts of post-confluent cells also had significantly higher rates of cleaved caspase 3 than pre-confluent grafts (20.2%±4.3% vs. 7.8%±1.8%, p<0.001). CONCLUSION: Pre-confluent cells should be used to maximize graft cell viability

A perspective on SIDS pathogenesis. The hypotheses: plausibility and evidence

Several theories of the underlying mechanisms of Sudden Infant Death Syndrome (SIDS) have been proposed. These theories have born relatively narrow beach-head research programs attracting generous research funding sustained for many years at expense to the public purse. This perspective endeavors to critically examine the evidence and bases of these theories and determine their plausibility; and questions whether or not a safe and reasoned hypothesis lies at their foundation. The Opinion sets specific criteria by asking the following questions: 1. Does the hypothesis take into account the key pathological findings in SIDS? 2. Is the hypothesis congruent with the key epidemiological risk factors? 3. Does it link 1 and 2? Falling short of any one of these answers, by inference, would imply insufficient grounds for a sustainable hypothesis. Some of the hypotheses overlap, for instance, notional respiratory failure may encompass apnea, prone sleep position, and asphyxia which may be seen to be linked to co-sleeping. For the purposes of this paper, each element will be assessed on the above criteria