Rebecca L Roberts

The Role of Neutrophils in the Production of Hypoxic-Ischemic Brain Injury in the Neonatal Rat

Neutrophils contribute to ischemic brain injury in adult animals. The role of neutrophils in perinatal hypoxic-ischemic (HI) brain injury is unknown. Allopurinol reduces neutrophil accumulation after tissue ischemia and is protective against HI brain injury. This study was designed to investigate how neutrophils contribute to perinatal hypoxic ischemic brain injury and how neutropenia compared with allopurinol in its neuroprotective effects. A HI insult was produced in the right cerebral hemisphere of 7-d-old rats by right common carotid artery ligation and systemic hypoxia. Half the rats were rendered neutropenic with an anti-neutrophil serum (ANS). At 15 min of recovery from hypoxia, half the neutropenic and nonneutropenic rats received allopurinol (135 mg/kg, s.c.). The protective effect of the four treatment combinations was determined on brain swelling at 42 h of recovery. Neutropenia reduced brain swelling by about 70%, p < 0.01. Allopurinol alone produced similar protection so that the relatively small number of animals studied did not permit assessment of an additive effect. Neutrophil accumulation in cerebral hemispheres was measured by myeloperoxidase (MPO) activity assay and by neutrophil counts in 6-μm sections stained by MPO and ANS immunostaining. MPO activity peaked between 4 and 8 h of recovery in both hemispheres. Hemispheric neutrophil counts peaked at the end of the HI insult and again at 18 h of recovery. Neutrophils were stained within blood vessels and did not infiltrate the injured brain before infarction had occurred. We conclude that neutrophils contribute to HI brain injury in the neonate and that neutrophil depletion before the insult is neuroprotective.

Allopurinol administered after inducing hypoxia-ischemia reduces brain injury in 7-day-old rats.

ABSTRACT: We determined that treatment of immature rats with allopurinol at 15 min after cerebral hypoxiaischemia reduces brain damage. Seven-d postnatal rats were subjected to right common carotid artery ligation followed by 2.25 h of hypoxia (8% O2). At 15 min of recovery in room air, the rat pups received either allopurinol (135 mg/kg s.c.) or saline. Some of the rats (n = 65) were killed at 42 h of recovery for measurement of cerebral hemispheric water content. Other animals (n = 63) were killed at 30 d for morphologic assessment of the severity of damage. In separate rats, we measured the levels of allopurinol and its metabolites in serum and in the brain around the time of peak serum levels. We also determined the effect of allopurinol on rat pup body temperature. Allopurinol reduced the increase in right hemisphere water content and markedly reduced atrophy. No cavitary lesions were seen in the 31 allopurinol-treated rats, whereas 15 of 32 saline-treated rats had cavitary cerebral lesions. Histologic examination confirmed that the allopurinol-treated rats had less brain injury. Serum allopurinol and oxypurinol peaked between 0.5 and 1 h after allopurinol injection. Their peak serum concentrations at 0.75 h postinjection combined was between 360 and 510 μM. Allopurinol did not lower rectal temperature more than 0.04°C. In conclusion, high-dose allopurinol administered at 15 min of recovery from cerebral hypoxia-ischemia markedly reduces both acute brain edema and long-term cerebral injury in immature rats.

Changes in iron histochemistry after hypoxic-ischemic brain injury in the neonatal rat

Iron can contribute to hypoxic‐ischemic brain damage by catalyzing the formation of free radicals. The immature brain has high iron levels and limited antioxidant defenses. The objective of this study was to describe the early alterations in nonheme iron histochemistry following a hypoxic‐ischemic (HI) insult to the brain of neonatal rats. We induced a HI insult to the right cerebral hemisphere in groups of 7‐day‐old rats. Rats were anesthetized, then their brains were perfused and fixed at 0, 1, 4, 8, 24 hr, and 1, 2, and 3 weeks of recovery. Forty‐micron‐thick frozen sections were stained for iron using the intensified Perls stain. Increased iron staining was first detected within the cytoplasm of cells with pyknotic nuclei at 4 hr of recovery. Staining increased rapidly over the first 24 hr in regions of ischemic injury. By 7days recovery, reactive glia and cortical blood vessels also stained. Increased staining in gray matter persisted at 3 weeks of recovery, whereas white matter tracts had fewer iron‐positive cells compared to normal. The early increase in iron staining could be caused by an accumulation of iron posthypoxic‐ischemic injury or a change in iron from nonstainable heme iron to stainable nonheme iron. Regardless of the source, our results indicate that there is an increase in iron available to promote oxidant stress in the neonatal rat brain following hypoxia‐ischemia. J. Neurosci. Res. 56:60–71, 1999. 

Differential expression of chemokines and chemokine receptors during microglial activation and inhibition

Intrauterine infection produces an inflammatory response in the fetus characterized by increased inflammatory cytokines in the fetal brain and activation of brain microglial cells. Intrauterine infection can release bacterial cell wall products into the fetal circulation. Lipopolysaccharides (LPS) are derived from the cell walls of gram negative organisms. The degree of microglial cell activation may influence the extent of brain injury following an inflammatory stimulus. Chemokines, which are released by activated microglia, regulate the influx of inflammatory cells to the brain. Accordingly, therapeutic strategies that reduce the extent of chemokine expression in microglial cells may prove neuroprotective. Minocycline (MN), a semisynthetic tetracycline derivative, protects brain against global and focal ischemia in rodents and inhibits microglial cell activation. To determine if minocycline can reduce the production of chemokines and chemokine receptors in response to LPS, microglial-like BV-2 and HAPI cells were cultured in the presence or absence of 100 ng/ml of LPS. Enzyme-linked immunosorbent assay (ELISA) and semi-quantitative RT-PCR were used to examine changes in inflammatory chemokines (macrophage inflammatory protein-1 (MIP-1alpha), regulated upon activation, normal T cell expressed and secreted (RANTES), and inducible protein-10 (IP-10)) and chemokine receptor (C-C chemokine receptor 5 (CCR5) and C-X-C chemokine receptor 3 (CXCR3)) production, respectively. We found that in both cell lines chemokine release after 4-, 8-, and 16-h exposure to LPS was significantly higher compared to non-exposed cells for all the chemokines measured, P<0.001. Minocycline inhibited chemokine release of LPS-stimulated BV-2 cells. There was even greater inhibition (up to 50%) of mRNA expression after exposure to LPS (P<0.001). We conclude that endotoxin enhanced the expression of chemokines and chemokine receptors in microglial-like cell lines. Modulation of this expression was achieved with minocycline. Recognition of the mechanisms whereby minocycline exerts its anti-inflammatory effect on microglia may uncover specific targets for pharmacologic intervention.

Timing of Neutrophil Depletion Influences Long-Term Neuroprotection in Neonatal Rat Hypoxic-Ischemic Brain Injury

In neonatal rats, neutrophils do not accumulate in ischemic brain parenchyma to the extent that they do in adult rodents. They are also confined to the intravascular compartment during the first few hours of recovery. However, neonatal rats rendered neutropenic have less brain swelling after a hypoxic-ischemic (HI) insult. In this study, we used the Rice-Vannucci model of HI brain injury in 7-d-old rats, and we depleted neutrophils before injury in one group and 4–8 h after injury in another group to determine 1) whether neutrophils contribute to cerebral atrophy, 2) whether neutropenia induced within 8 h after recovery from HI is neuroprotective, and 3) whether neutropenia preserved energy metabolites during the HI insult. Brain energy metabolites were measured at 0 h and 6 h of recovery. Brain atrophy was measured morphometrically on brain slices at 2 wk of recovery. In 67 rats, we found that neutropenia induced before the HI insult, but not after HI, reduced brain swelling at 42 h of recovery by about 75% (p < 0.001). In another 60 rats, we found that cerebral atrophy was reduced by 61% provided that neutropenia was induced before HI (p < 0.05). Total adenine nucleotides were better preserved in the neutropenic rats at the end of the HI insult (0 h recovery); p < 0.05. We conclude that neutrophils do contribute to vascular dysfunction either during the HI insult or early hours (<4–8 h) of recovery. Antineutrophil strategies initiated after this time are unlikely to be protective in the neonatal rat.

Minocycline modulates chemokine receptors but not interleukin-10 mRNA expression in hypoxic-ischemic neonatal rat brain.

Hypoxic‐ischemic (HI) brain injury in the perinatal period causes significant morbidity. Minocycline (MN) is a tetracycline derivative that has reduced brain injury in various animal models of neurodegeneration, including perinatal ischemia. To determine whether MN can modulate the expression of chemokine receptors and interleukin‐10 (IL10) in a model of neonatal brain injury, we produced an HI insult to the right cerebral hemisphere (ipsilateral) of the 7‐day‐old rat (PD7) by right common carotid artery ligation and 2.25 hr of hypoxia in 8% oxygen. MN (45 mg/kg, i.p.) or vehicle (PBS) was injected twice: 2 days and immediately before the HI insult. At 0, 1, 3, and 24 hr and 14 days after HI, total RNA from the ipsilateral and contralateral (exposed to hypoxia only) hemispheres was extracted, reverse transcribed, and amplified with gene‐specific primers using a semiquantitative RT‐PCR for macrophage inflammatory protein‐1α), interferon‐inducible protein (IP‐10), C‐C chemokine receptor 5 (CCR5; MIP‐1α receptor), C‐X‐C chemokine receptor 3 (CXCR3; IP‐10 receptor), and IL10. We found that, in the ipsilateral hemisphere, a significant (P < 0.05) increase in MIP‐1α, IP‐10, CCR5, and CXCR3 mRNA levels was observed. MN treatment decreased mRNA levels for CCR5 and CXCR3. In contrast, the levels of antiinflammatory cytokine IL10 were markedly decreased as a result of HI insult. Treatment with MN, however, had no effect on IL10. We conclude that MN decreased proinflammatory chemokine receptor expression but had little or no influence on the expression of antiinflammatory cytokine IL10. These effects confirm the antiinflammatory effect of MN in neonatal HI brain injury.

Reduction of hypoxic-ischemic brain swelling in the neonatal rat with PAF antagonist WEB 2170: lack of long-term protection.

Platelet activating factor (PAF) is an inflammatory lipid mediator released by ischemic brain. Our objectives were to use an inhibitor of PAF that does not readily cross the blood-brain barrier, WEB 2170, to study the role of intravascular PAF on brain swelling and subsequent brain atrophy in a neonatal rat model of hypoxic-ischemic brain injury. We injured the right cerebral hemisphere of 7-d-old rats by ligating the right common carotid artery and exposing the rats to 8% oxygen for 2.25 h. Forty-two rats received saline or the PAF antagonist WEB 2170, 1 h before hypoxia. We found that WEB 2170 pretreatment reduced swelling by 64% (p = 0.003). In contrast, treatment immediately after hypoxic-ischemic injury did not reduce swelling. In two additional experiments involving 103 rats, we found that pretreatment or repeated doses of PAF antagonist before and after hypoxic-ischemic injury did not reduce atrophy. We also found that the brain-penetrating PAF antagonist, BN 52021, did not prevent atrophy in our Wistar rat model. In conclusion, we were unable to reduce long-term brain injury with either PAF antagonist. WEB 2170 pretreatment reduced brain swelling by 64% without reducing atrophy. This suggests that although brain swelling may accompany cerebral infarction, it does not contribute to the pathogenesis of infarction and subsequent atrophy in the neonatal rat. The ability to reduce early postischemic brain swelling without reducing atrophy may be particularly unique to the immature animal with a compliant skull.

REDUCTION OF HYPOXIC ISCHEMIC BRAIN INJURY IN THE NEONATAL RAT WITH PAF ANTAGONIST WEB2170. † 2274

REDUCTION OF HYPOXIC ISCHEMIC BRAIN INJURY IN THE NEONATAL RAT WITH PAF ANTAGONIST WEB2170. † 2274

DELAYED ADMINISTRATION OF ALLOPURINOL AFTER CEREBRAL HYPOXIA-ISCHEMIA REDUCES BRAIN INJURY IN NEONATAL RATS |[dagger]| 1750

We have shown that Allopurinol (Allo) can reduce hypoxic-ischemic (HI) brain injury in 7 day old (P7) rats even when Allo (135mg/kg) is injected s.c. 15 min after termination of the insult. The post resuscitation time window during which Allo can reduce brain injury has not been explored. Objective: To determine if delayed administration of Allo, up to 4h after a HI insult to the brain of the P7 rat, can reduce brain injury. We produced a HI insult to the R cerebral hemisphere of 80 P7 rats by permanent R common carotid artery ligation and exposure to 8% oxygen for 2.25h at 36.8C. The rats then recovered with their dams in room air. Twenty rats per group were treated with a single s.c. injection of Saline (at 30min of recovery) or Allo (135mg/kg) at 30min, 60min, or 120min of recovery from the insult. At 42h of recovery we decapitated the rats and calculated% R hemisphere swelling (wet-dry wt. method). The RH swelled 23.7±10.7% (Mean ±SD) in the Saline group; In the Allo 30min group 7.4±10.3%; in the Allo 60min group 10.6±13.8% and in the Allo 120min group 17.5±8.7%, P = <0.001(Allo 30 and 60 vs Saline). In 44 other rats we produced an identical cerebral insult and treated with a single dose of Saline or Allo (135mg/kg) s.c. at 1 or 4h of recovery. At 14d recovery the rats were decapitated, the brains removed and immersion fixed. From three 2mm thick consistently positioned coronal sections through the damaged region of each brain, the average loss of area of the damaged R vs LH was expressed as%RH atrophy: RH atrophy was 41.1±18.2% (Mean ±SD) in the Saline treated group (n=15). In the Allo 1h group (n=13), it was 23.0±14.9% and in the Allo 4h group(n=16) it was 22.8±15.2%, P = <0.01 (Allo 1h and 4h groups vs Saline). Conclusion: A single dose of Allo (135mg/kg) s.c. administered 4 hours after the HI insult, markedly reduced brain injury in neonatal rats. This study suggest that the time window for neuroprotective intervention following birth asphyxia may span hours.

DELAYED INHIBITION OF NITRIC OXIDE PRODUCTION REDUCES POST HYPOXIC-ISCHEMIC BRAIN INJURY IN NEONATAL RATS † 1752

DELAYED INHIBITION OF NITRIC OXIDE PRODUCTION REDUCES POST HYPOXIC-ISCHEMIC BRAIN INJURY IN NEONATAL RATS † 1752