A Rat Model of Mild Intrauterine Hypoperfusion with Microcoil Stenosis

Intrauterine hypoperfusion/ischemia is one of the major causes of intrauterine/fetal growth restriction, preterm birth, and low birth weight. Most studies ...

national cerebral and cardiovascular center

National Cerebral and Cardiovascular Center

Neonatal stroke occurs in approximately 1/4000 live births and results in life-long neurological impairments: e.g., cerebral palsy. Currently, there is no evidence-based specific treatment for neonates with stroke. Several studies have reported the benefits of umbilical cord blood (UCB) cell treatment in rodent models of neonatal brain injury. However, all of the studies examined the effects of administering either the UCB mononuclear cell fraction or UCB-derived mesenchymal stem cells in neonatal rat models. The objective of this study was to examine the effects of human UCB CD34(+) cells (hematopoietic stem cell/endothelial progenitor cells) in a mouse model of neonatal stroke, which we recently developed. On postnatal day 12, immunocompromized (SCID) mice underwent permanent occlusion of the left middle cerebral artery (MCAO). Forty-eight hours after MCAO, human UCB CD34(+) cells (1×10(5)cells) were injected intravenously into the mice. The area in which cerebral blood flow (CBF) was maintained was temporarily larger in the cell-treated group than in the phosphate-buffered saline (PBS)-treated group at 24h after treatment. With cell treatment, the percent loss of ipsilateral hemispheric volume was significantly ameliorated (21.5±1.9%) compared with the PBS group (25.6±5.1%) when assessed at 7weeks after MCAO. The cell-treated group did not exhibit significant differences from the PBS group in either rotarod (238±46s in the sham-surgery group, 175±49s in the PBS group, 203±54s in the cell-treated group) or open-field tests. The intravenous administration of human UCB CD34(+) cells modestly reduced histological ischemic brain damage after neonatal stroke in mice, with a transient augmentation of CBF in the peri-infarct area.

Effects of intravenous administration of umbilical cord blood CD34(+) cells in a mouse model of neonatal stroke.

Much effort and many resources are being devoted to rehabilitative programs for children with disabilities caused by neonatal hypoxic-ischemic encephalopathy without clear evidence of the efficacy of such programs. We recently reported that rehabilitative training tasks during adolescence improve spatial learning impairment following neonatal hypoxic-ischemic injury in rats without histological improvement. In the present study we focused on sex differences. Wister rat pups were exposed to a unilateral hypoxic-ischemic insult at 7 days of age. Six weeks after hypoxia-ischemia, rehabilitative training tasks were started. The tasks consisted of the plus maze, the eight-arm radial maze, and the choice reaction time task. Sixteen weeks after the insult, the water maze task was performed to evaluate spatial learning ability. Afterwards, we morphologically examined brain injury. Our rehabilitative training significantly improved swimming time and length in females (P<0.01) but not in males. Likewise, the training ameliorated infarct areas in the injured cerebral hemisphere in females but not in males (P<0.01). These results suggest that it may be important to develop and evaluate cognitive rehabilitation programs for children with brain injury on the basis of gender.

Sex differences in the benefits of rehabilitative training during adolescence following neonatal hypoxia-ischemia in rats.

Progesterone and its metabolite, allopregnanolone, are neurosteroids that are present at high concentrations in fetal brains that decrease right after birth. Allopregnanolone is a potent positive modulator of γ-aminobutyric acid A (GABA(A)) receptor function. We examined the effect of exogenous administration of these steroids on hypoxic-ischemic encephalopathy in immature rats. Progesterone (10mg/kg), allopregnanolone (10mg/kg), or vehicle alone was intraperitoneally administered immediately before and then subcutaneously 6h and 24h after hypoxia-ischemia to postnatal day 7 (P7), day 14 (P14), and day 21 (P21) rats. The effects of the treatments were evaluated using histological analyses (hemispheric volumes and semi-quantitative scoring for neuropathologic injury). Both progesterone and allopregnanolone significantly exacerbated brain injury in P7 and P14 rats, but not in P21 rats. This detrimental effect was similar across the examined brain regions (the cortex, striatum, hippocampus, and thalamus) and showed no sex differences. Co-administration of the GABA(A) receptor antagonist, bicuculline, partially mitigated the exacerbating effect of allopregnanolone. Based on the similarity of the effects of these neurosteroids, we speculate that progesterone accentuates neuronal injury mainly via the activity of allopregnanolone. The present study indicates that the detrimental effects of allopregnanolone were, at least in part, mediated via GABAergic neuroexcitability. This is in line with the notion that GABA is excitatory for immature neurons, while it is inhibitory for mature neurons.

Progesterone and allopregnanolone exacerbate hypoxic-ischemic brain injury in immature rats.

Children with severe neonatal hypoxic-ischemic encephalopathy (HIE) die or develop life-long neurological impairments such as cerebral palsy and mental retardation. Decreased regional cerebral blood flow (CBF) is believed to be the predominant factor that determines the level of tissue injury in the immature brain. However, the spatio-temporal profiles of CBF after neonatal HIE are not well understood. CB17 mouse and Wistar rat pups were exposed to a unilateral hypoxic-ischemic (HI) insult at eight or seven days of age. Laser speckle imaging sequentially measured the cortical surface CBF before the hypoxic exposure and until 24h after the hypoxic exposure. Seven days after the HI insult, brain damage was morphologically assessed by measuring the hemispheric volumes and by semi-quantitative scoring for neuropathologic injury. The mean CBF on the ipsilateral hemisphere in mice decreased after carotid artery ligation. After the end of hypoxic insult (i.e., the reperfusion phase), the mean CBF level gradually rose and nearly attained its pre-surgery level by 9h of reperfusion. It then decreased. The degree of reduced CBF during reperfusion was well correlated with the degree of later morphological brain damage. The correlation was the strongest when the CBF was measured in the ischemic core region at 24h of reperfusion in mice (R²=0.89). A similar trend in results was found in rats. These results suggest that the CBF level during reperfusion may be a useful predictive factor for later brain damage in immature mice. This may enable optimizing brain damage for detail analyses.

Cerebral blood flow during reperfusion predicts later brain damage in a mouse and a rat model of neonatal hypoxic-ischemic encephalopathy.

Neonatal stroke occurs in 1/4000 live births and leaves life-long neurological impairments, such as cerebral palsy and epilepsy. Currently, the rodent models of neonatal stroke that are available exhibit significant inter-animal variability, which makes it difficult to accurately assess the mechanisms of brain injury and the efficacy of candidate treatments. We aimed to introduce a novel, highly reproducible model of stroke, middle cerebral artery occlusion (MCAO), in immature mice, and to evaluate the reproducibility of this model compared with a conventional hypoxia-ischemia (HI) model. Postnatal day 12 CB-17 mice underwent left MCAO by direct electrocoagulation. The MCAO model exhibited excellent long-term survival; 85% up to 8 weeks after the insult. Infarct was evident in every animal with MCAO (n=27) and was confined to the cortex, with the exception of some mild thalamic injury. While the % stroke volume 48 h after the insult was consistent in the MCAO group, range: 17.8-30.4% (minimum-maximum), it was substantially less consistent in the HI group, range: 3.0-70.1%. This contrasting variability between the two models was also evident in the cerebral blood flow, 24h after the insult, and in the ipsilateral hemispheric volume, as assessed at 8 weeks after the insult. Mice with MCAO exhibited significant neurofunctional deficits in the rotarod and open-field tests. Preclinical studies for neonatal stroke could become more reliable using this model, with even a potential reduction in the number of pups required for statistical significance. The contrasting variability between the two models may provide insights into the factors that contribute to inter-animal variability in brain injury.

A novel reproducible model of neonatal stroke in mice: comparison with a hypoxia-ischemia model.

Combinatorial therapy directed at both vascular and neurodegenerative aspects of dementia may offer a promising strategy for treatment of dementia, which often has a multifactorial basis in the elderly. We investigated whether the phosphodiesterase III inhibitor cilostazol, which is often used in the prevention of stroke and peripheral artery disease, may delay cognitive decline in the elderly receiving donepezil.

Cilostazol add-on therapy in patients with mild dementia receiving donepezil: a retrospective study.

Most therapeutic agents are administered intravenously (IV) in clinical settings and intraperitoneally (IP) in preclinical studies with neonatal rodents; however, it remains unclear whether intraperitoneal (IP) injection is truly an acceptable alternative for intravenous (IV) injection in preclinical studies. The objective of our study is to clarify the differences in the therapeutic effects of drugs and in the distribution of infused cells after an IP or IV injection in animals with brain injury.

Intraperitoneal and intravenous deliveries are not comparable in terms of drug efficacy and cell distribution in neonatal mice with hypoxia-ischemia.

We have recently established a novel mouse model of bilateral common carotid artery gradual occlusion. This model serves as a mimic of severe carotid artery disease with multiple cerebral infarctions induced by cerebrovascular insufficiency. In this study, we examined whether locomotor and cognitive impairment was induced in these mice using a test battery for neurological and cognitive functions. Adult C57BL/6J male mice were subjected to either ameroid constrictor (AC) placement to gradually narrow the bilateral common carotid arteries or to sham surgery. At 28 days post-surgery, locomotor activity was assessed by rotarod and wire hang tests, and cognitive function was assessed using the Y-maze and Morris water maze tests. Rotarod and wire hang tests showed a significantly shorter latency to fall in mice subjected to the placement of ACs compared with sham surgery mice. AC-implanted mice showed significant impairments in working memory on the Y-maze test and in spatial learning and reference memory on the Morris water maze test. Therefore, the current mouse model with AC placement on the bilateral common carotid arteries showed locomotor disability, learning deficits, and memory impairment, which well-replicated the outcomes of patients with ischemic carotid artery disease. This model will be useful for investigating the mechanisms underlying the neurological and cognitive deficits following cerebrovascular insufficiency and for exploring pharmacological interventions for stroke patients with severe carotid artery disease.

Motor and cognitive impairment in a mouse model of ischemic carotid artery disease.

Subcortical white matter (WM) is a frequent target of ischemic injury and extensive WM lesions are important substrates of vascular cognitive impairment (VCI) in humans. However, ischemic stroke rodent models have been shown to mainly induce cerebral infarcts in the gray matter, while cerebral hypoperfusion models show only WM rarefaction without infarcts. The lack of animal models consistently replicating WM infarct damage may partially explain why many neuroprotective drugs for ischemic stroke or VCI have failed clinically, despite earlier success in preclinical experiments. Here, we report a novel animal model of WM infarct damage with cognitive impairment can be generated by surgical implantation of different devices to the right and left common carotid artery (CCA) in C57BL/6J mice. Implantation of an ameroid constrictor to the right CCA resulted in gradual occlusion of the vessel over 28 d, whereas placement of a microcoil to the left CCA induced ∼50% arterial stenosis. Arterial spin labeling showed a gradual reduction of cerebral blood flow over 28 d post operation. Such reductions were more marked in the right, compared with the left, hemisphere and in subcortical, rather than the cortical, areas. Histopathological analysis showed multiple infarct damage in right subcortical regions, including the corpus callosum, internal capsule, hippocampal fimbria, and caudoputamen, in 81% of mice. Mice displaying such damage performed significantly poorer in locomotor and cognitive tests. The current mouse model replicates the phenotypes of human subcortical VCI, including multiple WM infarcts with motor and cognitive impairment.

A novel mouse model of subcortical infarcts with dementia.

Existing rodent models of vascular cognitive impairment (VCI) show abrupt changes in cerebral blood flow (CBF) and do not reliably replicate the clinical pathogenesis of VCI. We therefore aimed to develop a mouse model of VCI where CBF is gradually reduced, followed by subsequent progressive motor and cognitive impairment, after surgical intervention.

Gradual Carotid Artery Stenosis in Mice Closely Replicates Hypoperfusive Vascular Dementia in Humans.

Severe intrauterine ischemia is detrimental to the developing brain. The impact of mild intrauterine hypoperfusion on neurological development, however, is still unclear. We induced mild intrauterine hypoperfusion in rats on embryonic day 17 via arterial stenosis with metal microcoils wrapped around the uterine and ovarian arteries. All pups were born with significantly decreased birth weights. Decreased gray and white matter areas were observed without obvious tissue damage. Pups presented delayed newborn reflexes, muscle weakness, and altered spontaneous activity. The levels of proteins indicative of inflammation and stress in the vasculature, i.e., RANTES, vWF, VEGF, and adiponectin, were upregulated in the placenta. The levels of mRNA for proteins associated with axon and astrocyte development were downregulated in fetal brains. The present study demonstrates that even mild intrauterine hypoperfusion can alter neurological development, which mimics the clinical signs and symptoms of children with neurodevelopmental disorders born prematurely or with intrauterine growth restriction.

Mild intrauterine hypoperfusion reproduces neurodevelopmental disorders observed in prematurity.

Several cell therapies have been explored as novel therapeutic strategies for neonatal encephalopathy because the benefits of current treatments are limited. We previously reported that intravenous administration of human umbilical cord blood (hUCB) CD34+ cells (hematopoietic stem cells/endothelial progenitor cells) at 48 h after insult exerts therapeutic effects in neonatal mice with stroke, i.e., permanent middle cerebral artery occlusion. Although neonatal stroke and hypoxic-ischemic encephalopathy (HIE) are grouped under the term "neonatal encephalopathy," their pathogenesis differs. However, little is known about the differences in the effects of the same treatment between these 2 diseases. In this study, we investigated whether the same treatment protocol exerts therapeutic effects in neonatal mice with HIE. The treatment significantly ameliorated the decreased cerebral blood flow in the ischemic penumbra. Although the cylinder and rotarod tests showed a trend of amelioration of behavioral impairments from the treatment, these were not statistically significant. Morphological brain injuries were not altered by treatment. The cell administration did not cause any adverse effects apart from hyperactivity in the open-field test. Some of these findings are consistent with the results obtained in our previous study using a stroke model, but others are not. This study suggests that the treatment protocol needs to be optimized for each pathological condition.

Masahiro Tsuji

Department of Regenerative Medicine and Tissue Engineering

national cerebral and cardiovascular center

A Rat Model of Mild Intrauterine Hypoperfusion with Microcoil Stenosis

Masahiro Tsuji

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Regenerative Medicine and Tissue Engineering

national cerebral and cardiovascular center

A Rat Model of Mild Intrauterine Hypoperfusion with Microcoil Stenosis

Department of Regenerative Medicine and Tissue Engineering