Therapeutic hypothermia and hypoxia-ischemia in the term-equivalent neonatal rat: characterization of a translational preclinical model.

BACKGROUND: Hypoxic-ischemic encephalopathy (HIE) is a major cause of morbidity in survivors. Therapeutic hypothermia (TH) is the only available intervention, but the protection is incomplete. Preclinical studies of HIE/TH in the rodent have relied on the postnatal day (P) 7 rat whose brain approximates a 32-36 wk gestation infant, less relevant for these studies. We propose that HIE and TH in the term-equivalent P10 rat will be more translational. METHODS: P10-11 rat pups were subjected to unilateral hypoxia-ischemia (HI) and 4 h recovery in normothermic (N) or hypothermic (TH) conditions. Brain damage was assessed longitudinally at 24 h, 2 wk, and 12 wk. Motor function was assessed with the beam walk; recognition memory was measured by novel object recognition. RESULTS: Neuroprotection with TH was apparent at 2 and 12 wk in both moderately and severely damaged animals. TH improved motor function in moderate, but not severe, damage. Impaired object recognition occurred with severe damage with no evidence of protection of TH. CONCLUSION: This adaptation of the immature rat model of HI provides a reproducible platform to further study HIE/TH in which individual animals are followed up longitudinally to provide a useful translational preclinical model.

In vivo Monitoring of Cerebral Hemodynamics in the Immature Rat: Effects of Hypoxia-Ischemia and Hypothermia.

BACKGROUND: Neonatal hypoxic-ischemic (HI) encephalopathy occurs in 1-4 per 1,000 live term births and can cause devastating neurodevelopmental disabilities. Currently, therapeutic hypothermia (TH) is the only treatment with proven efficacy. Since TH is associated with decreased cerebral metabolism and cerebral blood flow (CBF), it is important to assess CBF at the bedside. Diffuse correlation spectroscopy (DCS) has emerged as a promising optical modality to noninvasively assess an index of CBF (CBFi) in both humans and animals. In this initial descriptive study, we employ DCS to monitor the evolution of CBFi following HI with or without TH in immature rats. We investigate potential relationships between CBF and subsequent cerebral damage. METHODS: HI was induced on postnatal day 10 or 11 rat pups by right common carotid artery ligation followed by 60-70 min hypoxia (8% oxygen). After HI, the pups recovered for 4 h under hypothermia (HI-TH group, n = 23) or normothermia (HI-N group, n = 23). Bilateral measurements of hemispheric CBFi were made with DCS in unanesthetized animals at baseline, before HI, and 0, 1, 2, 3, 4, 5, and 24 h after HI. The animals were sacrificed at either 1 or 4 weeks, and brain injury was scored on an ordinal scale of 0-5 (0 = no injury). RESULTS: Carotid ligation caused moderate bilateral decreases in CBFi. Following HI, an initial hyperemia was observed that was more prominent in the contralateral hemisphere. After initiation of TH, CBFi dropped significantly below baseline levels and remained reduced for the duration of TH. In contrast, CBFi in the HI-N group was not significantly decreased from baseline levels. Reductions in CBFi after 4 h of TH were not associated with reduced damage at 1 or 4 weeks. However, elevated ipsilateral CBFi and ipsilateral-to-contralateral CBFi ratios at 24 h were associated with worse outcome at 1 week after HI. CONCLUSIONS: Both HI and TH alter CBFi, with significant differences in CBFi between hypothermic and normothermic groups after HI. CBFi may be a useful biomarker of subsequent cerebral damage.

Neonatal encephalopathy: pre-clinical studies in neuroprotection.

Neonatal encephalopathy resulting from HI (hypoxia-ischaemia) continues to be a significant cause of mortality and morbidity in infants and children, affecting 1-2/1000 live term births and up to 60% of pre-term births. In order to understand the pathophysiology of this insult, as well as design therapeutic interventions, it is important to establish a relevant animal model for pre-clinical studies. One of the most frequently used models of HI-induced brain damage in immature animals is the unilateral carotid ligation/hypoxia model, initially developed in our laboratory more than 30 years ago. The original model employed the postnatal day 7 rat, whose brain is representative of that of a late gestation, pre-term [32-36 weeks GA (gestational age)] human infant. We, and others, have employed this model to characterize the pathophysiological, biochemical/energetic and neuropathological events following HI, as well as the determination of the unique characteristics of the immature brain that define its vulnerability to, and outcome from, HI. In defining the cascade of events following HI, it has become possible to identify potential targets for intervention and neuroprotection. Currently, the only available therapeutic intervention for neonatal encephalopathy in the term asphyxiated infant is therapeutic hypothermia, although this must be initiated within 6 h of birth and is at best partially effective in moderately injured infants. Ongoing pre-clinical studies are necessary to determine the basis for the partial protection afforded by hypothermia as well as the design of adjunct therapies to improve the outcome. The present review highlights the importance of using a well-characterized and relevant animal model to continue to pursue translational research in neuroprotection for the infant brain.

Mast cell isolation from the immature rat brain.

Mast cells are immune cells of hematopoietic origin that circulate as precursor cells prior to migration into vascularized tissues where they mature and undergo terminal differentiation in response to different cytokines within the local environment. Mast cells are well known as important regulators of inflammatory processes in peripheral tissues and recent studies support the involvement of mast cells in mediating the inflammatory response to cerebral hypoxia-ischemia in both the neonatal and adult brain. To better study mast cell function in vivo, it is important to be able to identify their environment-specific phenotype, as well as to study their interaction with other neural cells in vitro. Previous such studies of mast cells have relied on mast cells isolated from gut or bone marrow, or on a number of mast cell lines, all of which may behave differently from brain mast cells. The purpose of this study was to develop a technique for the isolation of mast cells from neonatal rat brain and to characterize these cells following hypoxia and hypoxia-ischemia. We adapted a previously described technique of coupling an antibody to the mast cell-specific FcεR1 receptor to a MACS microbead for the selective removal of intact mast cells from a neonatal brain preparation. We have isolated toluidine blue-positive brain mast cells that provide substrate for both protein analysis and in vitro studies. These cells express proteins previously used to specifically identify microglia in the brain, Iba-1 and coronin-1a. A subpopulation of mast cells in vivo also expresses Iba-1. Thus, we report a novel method for isolation of brain mast cells suitable for the study of mast cell phenotype under a variety of conditions. Further, we suggest that the use of proteins such as Iba-1 for the identification of microglia in the brain includes the caveat that mast cells may also be detected.

Increased cerebral matrix metalloprotease-9 activity is associated with compromised recovery in the diabetic db/db mouse following a stroke.

Diabetes is a major risk factor of stroke and is associated with increased frequency of stroke and a poorer prognosis for recovery. In earlier studies we have utilized type 2 diabetic mouse models of stroke and demonstrated that diabetic db/db and ob/ob mice experience larger infarct volumes and impaired recovery associated with greater infiltration of macrophage following hypoxic-ischemic (H/I) insult than their heterozygous non-diabetic db/+ and ob/+ littermates. To obtain a better understanding of the pathogenesis of the impaired recovery, we have investigated the role of matrix metalloproteases and their endogenous inhibitors in the breakdown of the blood-brain barrier (BBB) following H/I. Diabetic db/db mice showed a significant and more rapid increase in matrix metalloprotease (MMP)-9 mRNA, protein and gelatinolytic activity compared with db/+, which resulted in an increased degradation of occludin and collagen IV and subsequently, an increased BBB permeability and greater infiltration of neutrophils into the infarct area. The expression of the MMPs, especially in the db/+ mice, is preceded by an elevated expression of their endogenous tissue inhibitors of metalloproteases (TIMPs) 1, 2, and 3, whereas in the db/db mice, a lower expression of the TIMPs is associated with greater MMP 3 and 9 expression. These results suggest that an imbalance in the MMPs/TIMPs cascade in the diabetic mouse, particularly MMP-9, results in a greater neutrophil invasion, a compromised BBB and consequently a greater insult.

The PPAR-gamma agonist, darglitazone, restores acute inflammatory responses to cerebral hypoxia-ischemia in the diabetic ob/ob mouse.

Diabetes is an increased risk factor for stroke and results in increased brain damage in experimental animals and humans. The precise mechanisms are unclear, but our earlier studies in the db/db mice suggested that the cerebral inflammatory response initiating recovery was both delayed and diminished in the diabetic mice compared with the nondiabetic db/+ mice. In this study, we investigated the actions of the peroxisome proliferator-activated receptor (PPAR)-gamma agonist darglitazone in treating diabetes and promoting recovery after a hypoxic-ischemic (H/I) insult in the diabetic ob/ob mouse. Male ob/+ and ob/ob mice received darglitazone (1 mg/kg) for 7 days before induction of H/I. Darglitazone restored euglycemia and normalized elevated corticosterone, triglycerides, and very-low-density lipoprotein levels. Darglitazone dramatically reduced the infarct size in the ob/ob mice at 24 h of recovery compared with the untreated group (30+/-13% to 3.3+/-1.6%, n=6 to 8) but did not show any significant effect in the ob/+ mice. Microglial and astrocytic activation monitored by cytokine expression (interleukin-1beta and tumor necrosis factor-alpha) and in situ hybridization studies (bfl1 and glial fibrillary acidic protein) suggest a biphasic inflammatory response, with darglitazone restoring the compromised proinflammatory response(s) in the diabetic mouse at 4 h but suppressing subsequent inflammatory responses at 8 and 24 h in both control and diabetic mice.